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Current Research and Scholarly Interests

Our research focuses on developmental pathways that regulate hematopoietic cell growth and differentiation and are disrupted in the course of neoplastic transformation, particularly in leukemias and lymphomas. We employ a variety of experimental systems for our studies ranging from molecular biology to transgenic and knockout mice. Our current interests are:

1) Characterize novel families of oncoproteins (Pbx and Meis) that dimerize with and regulate the DNA binding properties of Hox proteins. We are investigating how Pbx and Meis proteins contribute to the specificity of Hox function in development and how disruption of their activities leads to neoplasia.

2) We have discovered a group of oncoproteins that are implicated in long-term maintenance of gene expression through their effects on the state of chromatin. We are studying the role that normal chromatin structure plays in gene regulation in hematopoietic cells and how its disruption leads to altered development and cancer.

3) We are defining the properties of cancer stem cells that initiate and sustain the unique disease features of acute leukemias through the use of various adoptive animal models.

Our studies have demonstrated that several of the proteins encoded by cellular oncogenes function in fundamental aspects of gene regulation. These are frequently activated by fusion to other transcriptional proteins resulting in chimeric transcription factors. We are studying the effects and consequences of protein fusion on the transcriptional and transforming activities of these proteins using in vitro and animal models.

In addition to these basic issues concerning leukemia pathogenesis, we are devising new diagnostic procedures for detecting and monitoring leukemia patients based on molecular genetic abnormalities in the malignant cells.

Abstract

Acute leukemias induced by MLL chimeric oncoproteins are among the subset of cancers distinguished by a paradoxical dependence on GSK-3 kinase activity for sustained proliferation. We demonstrate here that GSK-3 maintains the MLL leukemia stem cell transcriptional program by promoting the conditional association of CREB and its coactivators TORC and CBP with homedomain protein MEIS1, a critical component of the MLL-subordinate program, which in turn facilitates HOX-mediated transcription and transformation. This mechanism also applies to hematopoietic cells transformed by other HOX genes, including CDX2, which is highly expressed in a majority of acute myeloid leukemias, thus providing a molecular approach based on GSK-3 inhibitory strategies to target HOX-associated transcription in a broad spectrum of leukemias.

Abstract

The genetic programs that promote retention of self-renewing leukemia stem cells (LSCs) at the apex of cellular hierarchies in acute myeloid leukemia (AML) are not known. In a mouse model of human AML, LSCs exhibit variable frequencies that correlate with the initiating MLL oncogene and are maintained in a self-renewing state by a transcriptional subprogram more akin to that of embryonic stem cells (ESCs) than to that of adult stem cells. The transcription/chromatin regulatory factors Myb, Hmgb3, and Cbx5 are critical components of the program and suffice for Hoxa/Meis-independent immortalization of myeloid progenitors when coexpressed, establishing the cooperative and essential role of an ESC-like LSC maintenance program ancillary to the leukemia-initiating MLL/Hox/Meis program. Enriched expression of LSC maintenance and ESC-like program genes in normal myeloid progenitors and poor-prognosis human malignancies links the frequency of aberrantly self-renewing progenitor-like cancer stem cells (CSCs) to prognosis in human cancer.

Abstract

Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. Here we report pharmacological, physiological and genetic studies that demonstrate an oncogenic requirement for GSK3 in the maintenance of a specific subtype of poor prognosis human leukaemia, genetically defined by mutations of the MLL proto-oncogene. In contrast to its previously characterized roles in suppression of neoplasia-associated signalling pathways, GSK3 paradoxically supports MLL leukaemia cell proliferation and transformation by a mechanism that ultimately involves destabilization of the cyclin-dependent kinase inhibitor p27(Kip1). Inhibition of GSK3 in a preclinical murine model of MLL leukaemia provides promising evidence of efficacy and earmarks GSK3 as a candidate cancer drug target.

Abstract

Menin displays the unique ability to either promote oncogenic function in the hematopoietic lineage or suppress tumorigenesis in the endocrine lineage; however, its molecular mechanism of action has not been defined. We demonstrate here that these discordant functions are unified by menin's ability to serve as a molecular adaptor that physically links the MLL (mixed-lineage leukemia) histone methyltransferase with LEDGF (lens epithelium-derived growth factor), a chromatin-associated protein previously implicated in leukemia, autoimmunity, and HIV-1 pathogenesis. LEDGF is required for both MLL-dependent transcription and leukemic transformation. Conversely, a subset of menin mutations in multiple endocrine neoplasia type 1 patients abrogate interaction with LEDGF while preserving MLL interaction but nevertheless compromise MLL/menin-dependent functions. Thus, LEDGF critically associates with MLL and menin at the nexus of transcriptional pathways that are recurrently targeted in diverse diseases.

Abstract

Self-renewal is a defining characteristic of stem cells; however, the molecular pathways underlying its regulation are poorly understood. Here, we demonstrate that conditional inactivation of the Pbx1 proto-oncogene in the hematopoietic compartment results in a progressive loss of long-term hematopoietic stem cells (LT-HSCs) that is associated with concomitant reduction in their quiescence, leading to a defect in the maintenance of self-renewal as assessed by serial transplantation. Transcriptional profiling revealed that multiple stem cell maintenance factors are perturbed in Pbx1-deficient LT-HSCs, which prematurely express a large subset of genes, including cell-cycle regulators, normally expressed in non-self-renewing multipotent progenitors. A significant proportion of Pbx1-dependent genes is associated with the TGF-beta pathway, which serves a major role in maintaining HSC quiescence. Prospectively isolated, Pbx1-deficient LT-HSCs display altered transcriptional responses to TGF-beta stimulation in vitro, suggesting a possible mechanism through which Pbx1 maintenance of stem cell quiescence may in part be achieved.

Abstract

Using a mouse model of human acute myeloid leukemia (AML) induced by the MLL-AF9 oncogene, we demonstrate that colony-forming cells (CFCs) in the bone marrow and spleen of leukemic mice are also leukemia stem cells (LSCs). These self-renewing cells (1) are frequent, accounting for 25%-30% of myeloid lineage cells at late-stage disease; (2) generate a phenotypic, morphologic, and functional leukemia cell hierarchy; (3) express mature myeloid lineage-specific antigens; and (4) exhibit altered microenvironmental interactions by comparison with the oncogene-immortalized CFCs that initiated the disease. Therefore, the LSCs responsible for sustaining, expanding, and regenerating MLL-AF9 AML are downstream myeloid lineage cells, which have acquired an aberrant Hox-associated self-renewal program as well as other biologic features of hematopoietic stem cells.

Abstract

The Mixed-Lineage Leukemia (MLL) protein is a histone methyltransferase that is mutated in clinically and biologically distinctive subsets of acute leukemia. MLL normally associates with a cohort of highly conserved cofactors to form a macromolecular complex that includes menin, a product of the MEN1 tumor suppressor gene, which is mutated in heritable and sporadic endocrine tumors. We demonstrate here that oncogenic MLL fusion proteins retain an ability to stably associate with menin through a high-affinity, amino-terminal, conserved binding motif and that this interaction is required for the initiation of MLL-mediated leukemogenesis. Furthermore, menin is essential for maintenance of MLL-associated but not other oncogene induced myeloid transformation. Acute genetic ablation of menin reverses aberrant Hox gene expression mediated by MLL-menin promoter-associated complexes, and specifically abrogates the differentiation arrest and oncogenic properties of MLL-transformed leukemic blasts. These results demonstrate that a human oncoprotein is critically dependent on direct physical interaction with a tumor suppressor protein for its oncogenic activity, validate a potential target for molecular therapy, and suggest central roles for menin in altered epigenetic functions underlying the pathogenesis of hematopoietic cancers.

Abstract

Loss-of-function alterations of INK4A are commonly observed in lymphoid malignancies, but are consistently absent in pre-B cell leukemias induced by the chimeric oncoprotein E2a-Pbx1 created by t(1;19) chromosomal translocations. We report here that experimental induction of E2a-Pbx1 enhances expression of BMI-1, a lymphoid oncogene whose product functions as a transcriptional repressor of the INK4A-ARF tumor suppressor locus. Bmi-1-deficient hematopoietic progenitors are resistant to transformation by E2a-Pbx1; however, the requirement for Bmi-1 is alleviated in cells deficient for both Bmi-1 and INK4A-ARF. Furthermore, the adverse effects of E2a-Pbx1 on pre-B cell survival and differentiation are partially bypassed by forced expression of p16(Ink4a). These results link E2a-Pbx1 with Bmi-1 on an oncogenic pathway that is likely to play a role in the pathogenesis of human lymphoid leukemias through downregulation of the INK4A-ARF gene.

Abstract

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ?22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

Abstract

The zinc finger transcription factor Gli3 is an essential mediator of hedgehog signaling. Gli3 has a dynamic expression pattern during embryonic development. In the neural tube, Gli3 transcripts are patterned along the anteroposterior and dorsoventral axes such that the initial broad expression in the posterior neural tube becomes dorsally restricted as neurogenesis takes place. Little is known about the molecular mechanisms that regulate this dynamic expression. Here, we report on a phylogenetic analysis of the Gli3 locus that uncovered a novel regulatory element, HCNE1. HCNE1 contains a compound Pbx/Meis binding site that binds Pbx and Meis/Prep proteins in vitro and in vivo. We show that HCNE1 recapitulates Gli3 expression in the developing neural tube and that mutations in the Pbx/Meis binding site affect the spatiotemporal control of HCNE1 transcriptional activity. Ectopic expression or loss of function of Pbx and Meis/Prep proteins in the chick and mouse embryo results in aberrant expression of endogenous Gli3 transcripts. We propose a novel role for TALE proteins in establishing the correct spatiotemporal expression pattern of Gli3 in the vertebrate spinal cord, thus implicating TALE transcription factors in early embryonic patterning events controlled by Sonic hedgehog signaling.

Abstract

The MLL (mixed-lineage leukemia) proto-oncogene encodes a histone methyltransferase that creates the methylated histone H3K4 epigenetic marks, commonly associated with actively transcribed genes. In addition to its canonical histone methyltransferase SET domain, the MLL protein contains three plant homeodomain (PHD) fingers that are well conserved between species but whose potential roles and requirements for MLL function are unknown. Here, we demonstrate that the third PHD domain of MLL (PHD3) binds histone H3 trimethylated at lysine 4 (H3K4me3) with high affinity and specificity and H3K4me2 with 8-fold lower affinity. Biochemical and structural analyses using NMR and fluorescence spectroscopy identified key amino acids essential for the interaction with H3K4me3. Site-directed mutations of the residues involved in recognition of H3K4me3 compromised in vitro H3K4me3 binding but not in vivo localization of full-length MLL to chromatin sites in target promoters of MEIS1 and HOXA genes. Whereas intact PHD3 finger was necessary for MLL occupancy at these promoters, H3K4me3 binding was critical for MLL transcriptional activity. These results demonstrate that MLL occupancy and target gene activation can be functionally separated. Furthermore, these findings reveal that MLL not only "writes" the H3K4me3 mark but also binds the mark, and this binding is required for the transcriptional maintenance functions of MLL.

Abstract

MLL is a common target for chromosomal translocations associated with acute leukemia resulting in its fusion with a large variety of nuclear or cytoplasmic proteins that may activate its oncogenic properties by distinct but poorly understood mechanisms. The MLL-AF6 fusion gene represents the most common leukemogenic fusion of mixed lineage leukemia (MLL) to a cytoplasmic partner protein. Here, we identified a highly conserved Ras association (RA1) domain at the amino-terminus of AF6 as the minimal region sufficient for MLL-AF6 mediated myeloid progenitor immortalization in vitro and short latency leukemogenesis in vivo. Moreover, the ability of RA1 to activate MLL oncogenesis is conserved with its Drosophila ortholog, Canoe. Although the AF6 RA1 domain has previously been defined as an interaction surface for guanosine triphosphate-bound Ras, single amino acid substitutions known to abolish the AF6-Ras interaction did not abrogate MLL-AF6-mediated oncogenesis. Furthermore, fusion of MLL to heterologous RA domains of c-Raf1 or RalGDS, or direct fusion of MLL to constitutively active K-RAS, H-RAS, or RAP1 was not sufficient for oncogenic activation of MLL. Rather, the AF6 RA1 domain efficiently mediated self-association, suggesting that constitutive MLL self-association is a more common pathogenic mechanism for MLL oncogenesis than indicated by previous studies of rare MLL fusion partners.

Abstract

Despite advances in defining the critical molecular determinants for leukemia stem cell (LSC) generation and maintenance, little is known about the roles of microRNAs in LSC biology. Here, we identify microRNAs that are differentially expressed in LSC-enriched cell fractions (c-kit(+)) in a mouse model of MLL leukemia. Members of the miR-17 family were notably more abundant in LSCs compared with their normal counterpart granulocyte-macrophage progenitors and myeloblast precursors. Expression of miR-17 family microRNAs was substantially reduced concomitant with leukemia cell differentiation and loss of self-renewal, whereas forced expression of a polycistron construct encoding miR-17-19b miRNAs significantly shortened the latency for MLL leukemia development. Leukemias expressing increased levels of the miR-17-19b construct displayed a higher frequency of LSCs, more stringent block of differentiation, and enhanced proliferation associated with reduced expression of p21, a cyclin-dependent kinase inhibitor previously implicated as a direct target of miR-17 microRNAs. Knockdown of p21 in MLL-transformed cells phenocopied the overexpression of the miR-17 polycistron, including a significant decrease in leukemia latency, validating p21 as a biologically relevant and direct in vivo target of the miR-17 polycistron in MLL leukemia. Expression of c-myc, a crucial upstream regulator of the miR-17 polycistron, correlated with miR-17-92 levels, enhanced self-renewal, and LSC potential. Thus, microRNAs quantitatively regulate LSC self-renewal in MLL-associated leukemia in part by modulating the expression of p21, a known regulator of normal stem cell function.

Abstract

The Pbx TALE (three-amino-acid loop extension) homeodomain proteins interact with class 1 Hox proteins, which are master regulators of cell fate decisions. This study was performed to elucidate the role of the Pbx1 TALE protein in the corneal epithelium of mice.Pbx1(f/f) mice were crossed with mice containing Cre recombinase under the control of the K14 promoter. Subsequently, the eyes of these mice were dissected and prepared for histologic or molecular analysis.Tissue-specific deletion of Pbx1 in the corneal epithelium of mice resulted in corneal dystrophy and clouding that was apparent in newborns and progressively worsened with age. Thickening of the cornea epithelium was accompanied by stromal infiltration with atypical basal cells, severe disorganization of stromal collagen matrix, and loss of corneal barrier function. High epithelial cell turnover was associated with perturbed expression of developmental regulators and aberrant differentiation, suggesting an important function for Pbx1 in determining corneal identity.These studies establish an essential role of the Pbx1 proto-oncogene in corneal morphogenesis.

Abstract

Leukaemia stem cells (LSCs) are responsible for sustaining and propagating malignant disease, and, as such, are promising targets for therapy. Studies of human LSCs have served an important role in defining the major tenets of the cancer stem cell model, which centre on the frequencies of cancer stem cells, their potential hierarchical organisation and their degree of maturation. LSCs in acute myeloid leukaemia (AML) have recently been studied using mouse syngeneic models of leukaemia induced by MLL oncogenes. These studies have revealed that LSCs are more analogous to progenitor cells and employ embryonic stem cell-like genetic programmes for their maintenance, prompting a refinement of the original cancer stem cell model with important implications for design of therapies to selectively target LSCs.

Abstract

Treatment of hematologic malignancies is evolving from a uniform approach to targeted therapies directed at the underlying molecular abnormalities of disease. The mixed lineage leukemia (MLL) proto-oncogene is a recurrent site of genetic rearrangements in acute leukemias; and since its discovery in 1992, many advances have been made in understanding its role in leukemogenesis. A variety of MLL translocation partners have been described, and detailed structure/function studies have identified functional domains that are required for transformation. Proteins associated with the MLL core complex or its fusion partners have been isolated and characterized for their critical roles in leukemia pathogenesis. Downstream mediators of MLL transcriptional regulation and multiple collaborating signaling pathways have been described and characterized. These advances in our understanding of MLL-related leukemogenesis provide a foundation for ongoing and future efforts to develop novel therapeutic strategies that will hopefully result in better treatment outcomes.

Abstract

The patterning of the cardiovascular system into systemic and pulmonic circulations is a complex morphogenetic process, the failure of which results in clinically important congenital defects. This process involves extensive vascular remodeling and coordinated division of the cardiac outflow tract (OFT). We demonstrate that the homeodomain transcription factor Pbx1 orchestrates separate transcriptional pathways to control great-artery patterning and cardiac OFT septation in mice. Pbx1-null embryos display anomalous great arteries owing to a failure to establish the initial complement of branchial arch arteries in the caudal pharyngeal region. Pbx1 deficiency also results in the failure of cardiac OFT septation. Pbx1-null embryos lose a transient burst of Pax3 expression in premigratory cardiac neural crest cells (NCCs) that ultimately specifies cardiac NCC function for OFT development, but does not regulate NCC migration to the heart. We show that Pbx1 directly activates Pax3, leading to repression of its target gene Msx2 in NCCs. Compound Msx2/Pbx1-null embryos display significant rescue of cardiac septation, demonstrating that disruption of this Pbx1-Pax3-Msx2 regulatory pathway partially underlies the OFT defects in Pbx1-null mice. Conversely, the great-artery anomalies of compound Msx2/Pbx1-null embryos remain within the same spectrum as those of Pbx1-null embryos. Thus, Pbx1 makes a crucial contribution to distinct regulatory pathways in cardiovascular development.

Abstract

Congenital heart diseases are traditionally considered to be multifactorial in pathogenesis resulting from environmental and genetic interactions that determine penetrance and expressivity within a genetically predisposed family. Recent evidence suggests that genetic contributions have been significantly underestimated. However, single gene defects occur only in a minority of cases, and multigenetic causes of congenital heart diseases have not been fully demonstrated. Here, we show that interactions between alleles of 3 Pbx genes, which encode homeodomain transcription factors, are sufficient to determine the phenotypic presentation of congenital heart diseases in mice. A major role is served by Pbx1, whose inactivation results in persistent truncus arteriosus. Reduction or absence of Pbx2 or Pbx3 leads to Pbx1 haploinsufficiency and specific malformations that resemble tetralogy of Fallot, overriding aorta with ventricular septal defect, and bicuspid aortic valves. Disruption of Meis1, which encodes a Pbx DNA-binding partner, results in cardiac anomalies that resemble those caused by Pbx mutations. Each of the observed cardiac defects represents developmental abnormalities affecting distinct stages of cardiac outflow tract development and corresponds to specific types of human congenital heart disease. Thus, varied deficiencies in the Pbx gene family produce a full spectrum of cardiac defects involving the outflow tract, providing a framework for determining multigenetic causes of congenital heart anomalies.

Abstract

Gastrointestinal stromal tumors (GIST) occur primarily in the wall of the intestine and are characterized by activating mutations in the receptor tyrosine kinases genes KIT or PDGFRA. The diagnosis of GIST relies heavily on the demonstration of KIT/CD117 protein expression by immunohistochemistry. However, KIT expression is absent in approximately 4% to 15% of GIST and this can complicate the diagnosis of GIST in patients who may benefit from treatment with receptor tyrosine kinase inhibitors. We previously identified DOG1/TMEM16A as a novel marker for GIST using a conventional rabbit antipeptide antiserum and an in situ hybridization probe. Here, we describe 2 new monoclonal antibodies against DOG1 (DOG1.1 and DOG1.3) and compare their staining profiles with KIT and CD34 antibodies on 447 cases of GIST. These included 306 cases with known mutational status for KIT and PDGFRA from a molecular consultation service. In addition, 935 other mesenchymal tumors and 432 nonsarcomatous tumors were studied. Both DOG1 antibodies showed high sensitivity and specificity for GIST, with DOG1.1 showing some advantages. This antibody yielded positive staining in 370 of 425 (87%) scorable GIST, whereas CD117 was positive in 317 of 428 (74%) GIST and CD34 in 254 of 430 (59%) GIST. In GIST with mutations in PDGFRA, 79% (23/29) showed DOG1.1 immunoreactivity while only 9% (3/32) and 27% (9/33) stained for CD117 and CD34, respectively. Only 1 of 326 (0.3%) leiomyosarcomas and 1 of 39 (2.5%) synovial sarcomas among the 935 soft tissue tumors examined showed positive immunostaining for DOG1.1. In addition, DOG1.1 immunoreactivity was seen in fewer cases of carcinoma, melanoma, and seminoma as compared with KIT.

Abstract

Oncogenic mutations of the MLL histone methyltransferase confer an unusual ability to transform non-self-renewing myeloid progenitors into leukemia stem cells (LSCs) by mechanisms that remain poorly defined. Misregulation of Hox genes is likely to be critical for LSC induction and maintenance but alone it does not recapitulate the phenotype and biology of MLL leukemias, which are clinically heterogeneous--presumably reflecting differences in LSC biology and/or frequency. TALE (three-amino-acid loop extension) class homeodomain proteins of the Pbx and Meis families are also misexpressed in this context, and we thus employed knockout, knockdown, and dominant-negative genetic techniques to investigate the requirements and contributions of these factors in MLL oncoprotein-induced acute myeloid leukemia. Our studies show that induction and maintenance of MLL transformation requires Meis1 and is codependent on the redundant contributions of Pbx2 and Pbx3. Meis1 in particular serves a major role in establishing LSC potential, and determines LSC frequency by quantitatively regulating the extent of self-renewal, differentiation arrest, and cycling, as well as the rate of in vivo LSC generation from myeloid progenitors. Thus, TALE proteins are critical downstream effectors within an essential homeoprotein network that serves a rate-limiting regulatory role in MLL leukemogenesis.

Abstract

Pbx1, a homeodomain transcription factor that was originally identified as the product of a proto-oncogene in acute pre-B-cell leukemia, is a global regulator of embryonic development. However, embryonic lethality in its absence has prevented an assessment of its role in B-cell development. Here, using Rag1-deficient blastocyst complementation assays, we demonstrate that Pbx1 null embryonic stem (ES) cells fail to generate common lymphoid progenitors (CLPs) resulting in a complete lack of B and NK cells, and a partial impairment of T-cell development in chimeric mice. A critical role for Pbx1 was confirmed by rescue of B-cell development from CLPs following restoration of its expression in Pbx1-deficient ES cells. In adoptive transfer experiments, B-cell development from Pbx1-deficient fetal liver cells was also severely compromised, but not erased, since transient B lymphopoiesis was detected in Rag-deficient recipients. Conditional inactivation of Pbx1 in pro-B (CD19(+)) cells and thereafter revealed that Pbx1 is not necessary for B-cell development to proceed from the pro-B-cell stage. Thus, Pbx1 critically functions at a stage between hematopoietic stem cell development and B-cell commitment and, therefore, is one of the earliest-acting transcription factors that regulate de novo B-lineage lymphopoiesis.

Abstract

Improved understanding of the molecular pathways that suppress the genesis and maintenance of cancer stem cells will facilitate development of rationally targeted therapies. PU.1 is a transcription factor that is required for normal myelomonocytic differentiation in hematopoiesis, and reduced PU.1 activity has been associated with myeloid leukemogenesis in man and in mouse models. A recent study by Steidl et al. demonstrates that Junb and Jun, two AP-1 transcription factors, are critical downstream effectors of the tumor suppressor activity of PU.1, and that reduced expression of Junb, in particular, may be a common feature of acute myeloid leukemogenesis.

Abstract

The MLL gene is a frequent target for leukemia-associated chromosomal translocations that generate dominant-acting chimeric oncoproteins. These invariably contain the amino-terminal 1,400 residues of MLL fused with one of a variety of over 30 distinct nuclear or cytoplasmic partner proteins. Despite the consistent inclusion of the MLL amino-terminal region in leukemia oncoproteins, little is known regarding its molecular contributions to MLL-dependent oncogenesis. Using high-resolution mutagenesis, we identified three MLL domains that are essential for in vitro myeloid transformation via mechanisms that do not compromise subnuclear localization. These include the CXXC/Basic domain and two novel domains of unknown function. Point mutations in the CXXC domain that eliminate myeloid transformation by an MLL fusion protein also abolished recognition and binding of nonmethylated CpG DNA sites in vitro and transactivation in vivo. Our results define a critical role for the CXXC DNA binding domain in MLL-associated oncogenesis, most likely via epigenetic recognition of CpG DNA sites within the regulatory elements of target genes.

Abstract

Pbx proteins comprise a family of TALE (three amino acid loop extension) class homeodomain transcription factors that are implicated in developmental gene expression through their abilities to form hetero-oligomeric DNA-binding complexes and function as transcriptional regulators in numerous cell types. We demonstrate here that one member of this family, Pbx3, is expressed at high levels predominantly in the developing central nervous system, including a region of the medulla oblongata that is implicated in the control of respiration. Pbx3-deficient mice develop to term but die within a few hours of birth from central respiratory failure due to abnormal activity of inspiratory neurons in the medulla. This partially phenocopies the defect in mice deficient for Rnx, a metaHox homeodomain transcription factor, that we demonstrate here is capable of forming a DNA-binding complex with Pbx3. Rnx expression is unperturbed in Pbx3-deficient mice, but its ability to enhance transcription in vitro as a complex with TALE proteins is compromised in the absence of Pbx3. Thus, Pbx3 is essential for respiration and, like its DNA-binding partner Rnx, is critical for proper development of medullary respiratory control mechanisms. Pbx3-deficient mice provide a model for congenital central hypoventilation syndrome and suggest that Pbx3 mutations may promote the pathogenesis of this disorder.

Abstract

Forced dimerization or oligomerization has emerged as a powerful mechanism for unleashing the oncogenic properties of chimeric transcription factors in acute leukemias. Fusion of transcriptional regulators with a variety of heterologous partner proteins as a consequence of chromosomal rearrangements induces inappropriate self-association, leading to aberrant transcriptional properties and leukemogenesis. Forced dimerization/oligomerization may alter the association of a DNA-binding protein for its transcriptional cofactors, or the dimerization motifs themselves may constitutively recruit transcriptional effector molecules. Oligomerized chimeras may also sequester essential partners or cofactors to exert dominant-negative effects on target gene expression. A key mechanistic feature, and one with major clinical implications, is the nature of the transcriptional cofactors that are recruited by the dimerized oncoprotein. Chimeric RARalpha and acute myeloid leukemia 1 (AML1) proteins induce constitutive repression after the recruitment of corepressors, whereas inappropriate maintenance of target gene expression by mixed-lineage leukemia (MLL) chimeras may result from the recruitment of coactivators or the basal transcriptional machinery. Molecular therapies directed at enzymatic activities of the aberrantly recruited cofactors, or antagonism of dimerization itself, represent promising avenues of current and future investigation.

Abstract

MLL (for mixed-lineage leukemia) is a proto-oncogene that is mutated in a variety of human leukemias. Its product, a homolog of Drosophila melanogaster trithorax, displays intrinsic histone methyltransferase activity and functions genetically to maintain embryonic Hox gene expression. Here we report the biochemical purification of MLL and demonstrate that it associates with a cohort of proteins shared with the yeast and human SET1 histone methyltransferase complexes, including a homolog of Ash2, another Trx-G group protein. Two other members of the novel MLL complex identified here are host cell factor 1 (HCF-1), a transcriptional coregulator, and the related HCF-2, both of which specifically interact with a conserved binding motif in the MLL(N) (p300) subunit of MLL and provide a potential mechanism for regulating its antagonistic transcriptional properties. Menin, a product of the MEN1 tumor suppressor gene, is also a component of the 1-MDa MLL complex. Abrogation of menin expression phenocopies loss of MLL and reveals a critical role for menin in the maintenance of Hox gene expression. Oncogenic mutant forms of MLL retain an ability to interact with menin but not other identified complex components. These studies link the menin tumor suppressor protein with the MLL histone methyltransferase machinery, with implications for Hox gene expression in development and leukemia pathogenesis.

Abstract

Pbx2 is one of four mammalian genes that encode closely related TALE homeodomain proteins, which serve as DNA binding partners for a subset of Hox transcription factors. The expression and contributions of Pbx2 to mammalian development remain undefined, in contrast to the essential roles recently established for family members Pbx1 and Pbx3. Here we report that Pbx2 is widely expressed during embryonic development, particularly in neural and epithelial tissues during late gestation. Despite wide Pbx2 expression, mice homozygous mutant for Pbx2 are born at the expected Mendelian frequencies and exhibit no detectable abnormalities in development and organogenesis or reduction of long-term survival. The lack of an apparent phenotype in Pbx2(-)/(-) mice likely reflects functional redundancy, since the Pbx2 protein is present at considerably lower levels than comparable isoforms of Pbx1 and/or Pbx3 in embryonic tissues. In postnatal bone marrow and thymus, however, Pbx2 is the predominant high-molecular-weight (MW)-isoform Pbx protein detectable by immunoblotting. Nevertheless, the absence of Pbx2 has no measurable effect on steady-state hematopoiesis or immune function in adult mice, suggesting possible compensation by low-MW-isoform Pbx proteins present in these tissues. We conclude that the roles of Pbx2 in murine embryonic development, organogenesis, hematopoiesis, immune responses, and long-term survival are not essential.

Abstract

Differential expression of Hox genes is associated with normal hematopoiesis, whereas inappropriate maintenance of Hox gene expression, particularly Hoxa7 and Hoxa9, is a feature of leukemias harboring mixed-lineage leukemia (MLL) mutations. To understand the pathogenic roles of Hox genes in MLL leukemias, we assessed the impact of Hoxa7 or Hoxa9 nullizygosity on hematopoietic progenitor compartments and their susceptibility to MLL-induced leukemias. Selective reductions in the absolute numbers of committed progenitors, but not of hematopoietic stem cells, distinguished Hoxa7- and Hoxa9-deficient mice. Megakaryocytic/erythroid progenitor (MEP) reductions in Hoxa7(-/-) mice correlated with reticulocytosis and thrombocytopenia without anemia. Conversely, Hoxa9(-/-) mice displayed marked lymphopenia and substantial reductions of common lymphoid progenitors (CLPs) and lymphoid precursors, in addition to significant reductions of common myeloid progenitors (CMPs) and granulocyte/monocyte progenitors (GMPs). In retroviral transduction/transplantation assays, Hoxa7- and Hoxa9-deficient progenitors remained susceptible to transformation by MLL-GAS7, which activates MLL through a dimerization-dependent mechanism. However, Hoxa7(-/-) or Hoxa9(-/-) progenitors were less efficient in generating transformed blast colony-forming units (CFUs) in vitro and induced leukemias with longer disease latencies, reduced penetrance, and less mature phenotypes. Thus, Hoxa7 and Hoxa9 contribute to hematopoietic progenitor homeostasis but are not necessary for MLL-GAS7-mediated leukemogenesis, yet they appear to affect disease latency, penetrance, and phenotypes consistent with their critical roles as downstream targets of MLL fusion proteins.

Abstract

We have used the hematopoietic system as a model to investigate whether acute myeloid leukemia arises exclusively from self-renewing stem cells or also from short-lived myeloid progenitors. When transduced with a leukemogenic MLL fusion gene, prospectively isolated stem cells and myeloid progenitor populations with granulocyte/macrophage differentiation potential are efficiently immortalized in vitro and result in the rapid onset of acute myeloid leukemia with similar latencies following transplantation in vivo. Regardless of initiating cell, leukemias displayed immunophenotypes and gene expression profiles characteristic of maturation arrest at an identical late stage of myelomonocytic differentiation, putatively a monopotent monocytic progenitor stage. Our findings unequivocally establish the ability of transient repopulating progenitors to initiate myeloid leukemias in response to an MLL oncogene, and support the existence of cancer stem cells that do not necessarily overlap with multipotent stem cells of the tissue of origin.

Abstract

Transcriptional deregulation through the production of dominant-acting chimeric transcription factors derived from chromosomal translocations is a common theme in the pathogenesis of acute leukemias; however, the essential target genes for acute leukemogenesis are unknown. We demonstrate here that primary myeloid progenitors immortalized by various MLL oncoproteins exhibit a characteristic Hoxa gene cluster expression profile, which reflects that preferentially expressed in the myeloid clonogenic progenitor fraction of normal bone marrow. Continued maintenance of this MLL-dependent Hoxa gene expression profile is associated with conditional MLL-associated myeloid immortalization. Moreover, Hoxa7 and Hoxa9 were specifically required for efficient in vitro myeloid immortalization by an MLL fusion protein but not other leukemogenic fusion proteins. Finally, in a bone marrow transduction/transplantation model, Hoxa9 is essential for MLL-dependent leukemogenesis in vivo, a primary requirement detected at the earliest stages of disease initiation. Thus, a genetic reliance on Hoxa7 and Hoxa9 in MLL-mediated transformation demonstrates a gain-of-function mechanism for MLL oncoproteins as upstream constitutive activators that promote myeloid transformation via a Hox-dependent mechanism.

Abstract

MLL is a histone methyltransferase that can be converted into an oncoprotein by acquisition of transcriptional effector domains following heterologous protein fusions with a variety of nuclear transcription factors, cofactors, or chromatin remodeling proteins in acute leukemias. Here we demonstrate an alternative mechanism for activation of MLL following fusions with proteins (AF1p/Eps15 and GAS7) that normally reside in the cytoplasm. The coiled-coil oligomerization domains of these proteins are necessary and sufficient for leukemogenic transformation induced by the respective MLL fusion proteins. Furthermore, homodimerization of MLL by synthetic dimerization modules mimics bona fide MLL fusion proteins resulting in Hox gene activation and enhanced self-renewal of hematopoietic progenitors. Our studies support an oligomerization-dependent mechanism for oncogenic conversion of MLL, presumably in part by recruitment of accessory factors through the dimerized MLL moiety of the chimeric protein.

Genomewide demarcation of RNA polymerase II transcription units revealed by physical fractionation of chromatinPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICANagy, P. L., Cleary, M. L., Brown, P. O., Lieb, J. D.2003; 100 (11): 6364-6369

Abstract

Epigenetic modifications of chromatin serve an important role in regulating the expression and accessibility of genomic DNA. We report here a genomewide approach for fractionating yeast chromatin into two functionally distinct parts, one containing RNA polymerase II transcribed sequences, and the other comprising noncoding sequences and genes transcribed by RNA polymerases I and III. Noncoding regions could be further fractionated into promoters and segments lacking promoters. The observed separations were apparently based on differential crosslinking efficiency of chromatin in different genomic regions. The results reveal a genomewide molecular mechanism for marking promoters and genomic regions that have a license to be transcribed by RNA polymerase II, a previously unrecognized level of genomic complexity that may exist in all eukaryotes. Our approach has broad potential use as a tool for genome annotation and for the characterization of global changes in chromatin structure that accompany different genetic, environmental, and disease states.

Abstract

Pbx1 encodes a TALE homeodomain transcription factor that regulates developmental gene expression in a variety of tissues. Loss-of-function studies have demonstrated a critical role for Pbx1 in cellular proliferation and patterning and suggest its involvement in numerous regulatory pathways. In this study, examination of metanephric development in Pbx1(-/-) embryos was conducted to further elucidate Pbx1-dependent processes during organogenesis. Prior to death at E15.5, Pbx1(-/-) embryos displayed kidneys that were reduced in size, axially mispositioned, and in more severe cases, exhibited unilateral agenesis. Analysis with molecular markers revealed the effective induction of tubulogenic mesenchyme; however, Pbx1(-/-) kidneys contained fewer nephrons and were characterized by expanded regions of mesenchymal condensates in the nephrogenic zone. Despite the restricted expression of Pbx1 in metanephric mesenchyme, developing nephrons, and stroma, decreased branching and elongation of the ureter were also observed. Moreover, heterologous recombination studies with explant cultures verified that Pbx1(-/-) renal defects arose exclusively from mesenchymal dysfunction. Taken together, these data establish a role for Pbx1 in mesenchymal-epithelial signaling and demonstrate that Pbx1 is an essential regulator of mesenchymal function during renal morphogenesis.

Abstract

A specific association with mixed lineage leukemias suggests that MLL oncoproteins may selectively target early multipotent hematopoietic progenitors or stem cells. We demonstrate here that a representative MLL fusion protein, MLL-GAS7, impairs the differentiation and enhances the in vitro growth of murine hematopoietic cells with multipotent features. The multilineage differentiation potential of these cells was suggested by their immuno-phenotypes and transcriptional programs and confirmed by their ability to induce three pathologically distinct leukemias in mice, including an acute biphenotypic leukemia (ABL) that recapitulates the distinctive hallmark features of many MLL-associated leukemias in humans. This experimental modeling of ABL in mice highlights its origin from multipotential progenitors that arrest at a bipotential stage specifically targeted or induced by MLL oncogenes.

Abstract

The mixed lineage leukemia (MLL) gene undergoes fusions with a diverse set of genes as a consequence of chromosomal translocations in acute leukemias. Two of these partner genes code for members of the forkhead subfamily of transcription factors designated FKHRL1 and AFX. We demonstrate here that MLL-FKHRL1 enhances the self-renewal of murine myeloid progenitors in vitro and induces acute myeloid leukemias in syngeneic mice. The long latency (mean = 157 days), reduced penetrance, and hematologic features of the leukemias were very similar to those observed for the forkhead fusion protein MLL-AFX and contrasted with the more aggressive features of leukemias induced by MLL-AF10. Transformation mediated by MLL-forkhead fusion proteins required 2 conserved transcriptional effector domains (CR2 and CR3), each of which alone was not sufficient to activate MLL. A synthetic fusion of MLL with FKHR, a third mammalian forkhead family member that contains both effector domains, was also capable of transforming hematopoietic progenitors in vitro. A comparable requirement for 2 distinct transcriptional effector domains was also displayed by VP16, which required its proximal minimal transactivation domain (MTD/H1) and distal H2 domain to activate the oncogenic potential of MLL. The functional importance of CR2 was further demonstrated by its ability to substitute for H2 of VP16 in domain-swapping experiments to confer oncogenic activity on MLL. Our results, based on bona fide transcription factors as partners for MLL, unequivocally establish a transcriptional effector mechanism to activate its oncogenic potential and further support a role for fusion partners in determining pathologic features of the leukemia phenotype.

Associations between human disease genes and overlapping gene groups and multiple amino acid runsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAKarlin, S., CHEN, C. F., Gentles, A. J., Cleary, M.2002; 99 (26): 17008-17013

Abstract

Overlapping gene groups (OGGs) arise when exons of one gene are contained within the introns of another. Typically, the two overlapping genes are encoded on opposite DNA strands. OGGs are often associated with specific disease phenotypes. In this report, we identify genes with OGG architecture and genes encoding multiple long amino acid runs and examine their relations to diseases. OGGs appear to be susceptible to genomic rearrangements as happens commonly with the loci of the DiGeorge syndrome on human chromosome 22. We also examine the degree of conservation of OGGs between human and mouse. Our analyses suggest that (i) a high proportion of genes in OGG regions are disease-associated, (ii) genomic rearrangements are likely to occur within OGGs, possibly as a consequence of anomalous sequence features prevalent in these regions, and (iii) multiple amino acid runs are also frequently associated with pathologies.

Abstract

The chimeric transcription factor E2a-Hlf is an oncoprotein associated with a subset of acute lymphoblastic leukemias of early B-lineage derivation. We employed a retroviral transduction-transplantation approach to evaluate the oncogenic effects of E2a-Hlf on murine B-cell progenitors harvested from adult bone marrow. Expression of E2a-Hlf induced short-lived clusters of primary hematopoietic cells but no long-term growth on preformed bone marrow stromal cell layers comprised of the AC6.21 cell line. Coexpression with Bcl-2, however, resulted in the sustained self-renewal of early preB-I cells that required stromal and interleukin-7 (IL-7) support for growth in vitro. Immortalized cells were unable to induce leukemias after transplantation into nonirradiated syngeneic hosts, unlike the leukemic properties and cytokine independence of preB-I cells transformed by p190(Bcr-Abl) under identical in vitro conditions. However, bone marrow cells expressing E2a-Hlf in combination with Bcl-2, but not E2a-Hlf alone, induced leukemias in irradiated recipients with long latencies, demonstrating both a requirement for suppression of apoptosis and the need for further secondary mutations in leukemia pathogenesis. Coexpression of IL-7 substituted for Bcl-2 to induce the in vitro growth of pre-B cells expressing E2a-Hlf, but leukemic conversion required additional abrogation of undefined stromal requirements and was associated with alterations in the Arf/Mdm2/p53 pathway. Thus, E2a-Hlf enhances the self-renewal of bone marrow B-cell progenitors without inciting a p53 tumor surveillance response or abrogating stromal and cytokine requirements for growth, which are nevertheless abrogated during progression to a leukemogenic phenotype.

Abstract

MLL-AFX is a fusion gene created by t(X;11) chromosomal translocations in a subset of acute leukemias of either myeloid or lymphoid derivation. It codes for a chimeric protein consisting of MLL fused to AFX, a forkhead transcription factor that normally regulates genes involved in apoptosis and cell cycle progression. We demonstrate here that forced expression of MLL-AFX enhances the self-renewal of hematopoietic progenitors in vitro and induces acute myeloid leukemias after long latencies in syngeneic recipient mice. MLL-AFX interacts with the transcriptional coactivator CBP, which is also a fusion partner for MLL in human leukemias. A potent minimal transactivation domain (CR3) at the C terminus of AFX mediates interactions with the KIX domain of CBP and is necessary for transformation of myeloid progenitors by MLL-AFX. However, CR3 alone is not sufficient, suggesting that simple acquisition of a transactivation domain per se does not activate the oncogenic potential of MLL. Rather, two conserved transcriptional effector domains (CR2 and CR3) of AFX are required for full oncogenicity of MLL-AFX and also endow it with the potential to competitively interfere with transcription and apoptosis mediated by wild-type forkhead proteins. Furthermore, a dominant-negative mutant of AFX containing CR2 and CR3 enhances the growth of myeloid progenitors in vitro, although considerably less effectively than does MLL-AFX. Taken together, these data suggest that recruitment of transcriptional cofactors utilized by forkhead proteins is a critical requirement for oncogenic action of MLL-AFX, which may impact both MLL- and forkhead-dependent transcriptional pathways.

Abstract

Asexual development in Toxoplasma gondii is a vital aspect of the parasite's life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of approximately 4,400 Toxoplasma cDNAs, representing a minimum of approximately 600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein, SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.

Abstract

The t(10;11)(p12;q23) chromosomal translocation in human acute myeloid leukemia results in the fusion of the MLL and AF10 genes. The latter codes for a novel leucine zipper protein, one of many MLL fusion partners of unknown function. In this report, we demonstrate that retroviral-mediated transduction of an MLL-AF10 complementary DNA into primary murine myeloid progenitors enhanced their clonogenic potential in serial replating assays and led to their efficient immortalization at a primitive stage of myeloid differentiation. Furthermore, MLL-AF10-transduced cells rapidly induced acute myeloid leukemia in syngeneic or severe combined immunodeficiency recipient mice. Structure/function analysis showed that a highly conserved 82-amino acid portion of AF10, comprising 2 adjacent alpha-helical domains, was sufficient for immortalizing activity when fused to MLL. Neither helical domain alone mediated immortalization, and deletion of the 29-amino acid leucine zipper within this region completely abrogated transforming activity. Similarly, the minimal oncogenic domain of AF10 exhibited transcriptional activation properties when fused to the MLL or GAL4 DNA-binding domains, while neither helical domain alone did. However, transcriptional activation per se was not sufficient because a second activation domain of AF10 was neither required nor competent for transformation. The requirement for alpha-helical transcriptional effector domains is similar to the oncogenic contributions of unrelated MLL partners ENL and ELL, suggesting a general mechanism of myeloid leukemogenesis by a subset of MLL fusion proteins, possibly through specific recruitment of the transcriptional machinery.

Abstract

Pseudophosphatases display extensive sequence similarities to phosphatases but harbor amino acid alterations in their active-site consensus motifs that render them catalytically inactive. A potential role in substrate trapping or docking has been proposed, but the specific requirements for pseudophosphatases during development and differentiation are unknown. We demonstrate here that Sbf1, a pseudophosphatase of the myotubularin family, is expressed at high levels in seminiferous tubules of the testis, specifically in Sertoli's cells, spermatogonia, and pachytene spermatocytes, but not in postmeiotic round spermatids. Mice that are nullizygous for Sbf1 exhibit male infertility characterized by azoospermia. The onset of the spermatogenic defect occurs in the first wave of spermatogenesis at 17 days after birth during the synchronized progression of pachytene spermatocytes to haploid spermatids. Vacuolation of the Sertoli's cells is the earliest observed phenotype and is followed by reduced formation of spermatids and eventual depletion of the germ cell compartment in older mice. The nullizygous phenotype in conjunction with high-level expression of Sbf1 in premeiotic germ cells and Sertoli's cells is consistent with a crucial role for Sbf1 in transition from diploid to haploid spermatocytes. These studies demonstrate an essential role for a pseudophosphatase and implicate signaling pathways regulated by myotubularin family proteins in spermatogenesis and germ cell differentiation.

Abstract

Pbx1 is a member of the TALE (three-amino acid loop extension) class of homeodomain transcription factors, which are components of hetero-oligomeric protein complexes thought to regulate developmental gene expression and to maintain differentiated cell states. In vitro studies have shown that Pbx1 regulates the activity of Ipf1 (also known as Pdx1), a ParaHox homeodomain transcription factor required for the development and function of the pancreas in mice and humans. To investigate in vivo roles of Pbx1 in pancreatic development and function, we examined pancreatic Pbx1 expression, and morphogenesis, cell differentiation and function in mice deficient for Pbx1. Pbx1-/- embryos had pancreatic hypoplasia and marked defects in exocrine and endocrine cell differentiation prior to death at embryonic day (E) 15 or E16. In these embryos, expression of Isl1 and Atoh5, essential regulators of pancreatic morphogenesis and differentiation, was severely reduced. Pbx1+/- adults had pancreatic islet malformations, impaired glucose tolerance and hypoinsulinemia. Thus, Pbx1 is essential for normal pancreatic development and function. Analysis of trans-heterozygous Pbx1+/- Ipf1+/- mice revealed in vivo genetic interactions between Pbx1 and Ipf1 that are essential for postnatal pancreatic function; these mice developed age-dependent overt diabetes mellitus, unlike Pbx1+/- or Ipf1+/- mice. Mutations affecting the Ipf1 protein may promote diabetes mellitus in mice and humans. This study suggests that perturbation of Pbx1 activity may also promote susceptibility to diabetes mellitus.

A trithorax-group complex purified from Saccharomyces cerevisiae is required for methylation of histone H3PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICANagy, P. L., Griesenbeck, J., Kornberg, R. D., Cleary, M. L.2002; 99 (1): 90-94

Abstract

Histone methylation has emerged as an important mechanism for regulating the transcriptional accessibility of chromatin. Several methyltransferases have been shown to target histone amino-terminal tails and mark nucleosomes associated with either euchromatic or heterochromatic states. However, the biochemical machinery responsible for regulating histone methylation and integrating it with other cellular events has not been well characterized. We report here the purification, molecular identification, and genetic and biochemical characterization of the Set1 protein complex that is necessary for methylation of histone H3 at lysine residue 4 in Saccharomyces cerevisiae. The seven-member 363-kDa complex contains homologs of Drosophila melanogaster proteins Ash2 and Trithorax and Caenorhabditis elegans protein DPY-30, which are implicated in the maintenance of Hox gene expression and regulation of X chromosome dosage compensation, respectively. Mutations of Set1 protein comparable to those that disrupt developmental function of its Drosophila homolog Trithorax abrogate histone methylation in yeast. These studies suggest that epigenetic regulation of developmental and sex-specific gene expression are species-specific readouts for a common chromatin remodeling machinery associated mechanistically with histone methylation.

Abstract

Through sequencing projects and, more recently, array-based expression analysis experiments, a wealth of genetic data has become accessible via online resources. In contrast, few of the (molecular-) cytogenetic aberration data collected in the last decades are available in a format suitable for data mining procedures. www.progenetix.net is a new online repository for previously published chromosomal aberration data, allowing the addition of band-specific information about chromosomal imbalances to oncologic data analysis efforts.http://www.progenetix.netmbaudis@stanford.edu

Abstract

The MLL (Mixed Lineage Leukemia) gene is a common target for chromosomal translocations associated with human acute leukemias. These translocations result in a gain of MLL function by generating novel chimeric proteins containing the amino-terminus of MLL fused in-frame with one of 30 distinct partner proteins. Structure/function studies using an in vitro myeloid progenitor immortalization assay have revealed that at least four nuclear partner proteins contribute transcriptional effector properties to MLL to produce a range of chimeric transcription factors with leukemogenic potential. Mouse models suggest that expression of an MLL fusion protein is necessary but not sufficient for leukemogenesis. Interestingly, whilst all MLL fusion proteins tested so far phenocopy each other with respect to in vitro immortalization, the latency period required for the onset of acute leukemia in vivo is variable and partner protein dependent. We discuss potential mechanisms that may account for the ability of distinct MLL fusion proteins to promote short or long latency leukemogenesis.

Abstract

Pbx1 and a subset of homeodomain proteins collaboratively bind DNA as higher-order molecular complexes with unknown consequences for mammalian development. Pbx1 contributions were investigated through characterization of Pbx1-deficient mice. Pbx1 mutants died at embryonic day 15/16 with severe hypoplasia or aplasia of multiple organs and widespread patterning defects of the axial and appendicular skeleton. An obligatory role for Pbx1 in limb axis patterning was apparent from malformations of proximal skeletal elements, but distal structures were unaffected. In addition to multiple rib and vertebral malformations, neural crest cell-derived skeletal structures of the second branchial arch were morphologically transformed into elements reminiscent of first arch-derived cartilages. Although the skeletal malformations did not phenocopy single or compound Hox gene defects, they were restricted to domains specified by Hox proteins bearing Pbx dimerization motifs and unaccompanied by alterations in Hox gene expression. In affected domains of limbs and ribs, chondrocyte proliferation was markedly diminished and there was a notable increase of hypertrophic chondrocytes, accompanied by premature ossification of bone. The pattern of expression of genes known to regulate chondrocyte differentiation was not perturbed in Pbx1-deficient cartilage at early days of embryonic skeletogenesis, however precocious expression of Col1a1, a marker of bone formation, was found. These studies demonstrate a role for Pbx1 in multiple developmental programs and reveal a novel function in co-ordinating the extent and/or timing of proliferation with terminal differentiation. This impacts on the rate of endochondral ossification and bone formation and suggests a mechanistic basis for most of the observed skeletal malformations.

Abstract

Sbf1 (SET binding factor 1) is a pseudo-phosphatase related to the myotubularin family of dual specificity phosphatases, some of which have been implicated in cellular growth and differentiation by virtue of their mutation in human genetic disorders. Sbf1 contains germline-encoded alterations of its myotubularin homology domain that render it non-functional as a phosphatase. We report here the complete structure of Sbfl and further characterization of its growth regulatory properties. In addition to its similarity to myotubularin, the predicted full-length Sbf1 protein contains pleckstrin (PH) and GEF homology domains that are conserved in several proteins implicated in signaling and growth control. Forced expression of wild-type Sbfl in NIH 3T3 cells inhibited their proliferation and altered their morphology. These effects required intact PH, GEF and myotubularin homology domains, implying that growth inhibition may be an intrinsic property of wild-type Sbf1. Conversely, deletion of its conserved N-terminal 44 amino acids alone was sufficient to convert Sbf1 from an inhibitor of cellular growth to a transforming protein in NIH 3T3 cells. Oncogenic forms of Sbf1 partially localized to the nucleus, in contrast to the exclusively cytoplasmic subcellular localization of endogenous Sbf1 in all cell lines and mammalian tissues tested. These data show that the N-terminal GEF homology domain serves to inhibit the transforming effects of Sbf1, possibly sequestering the protein to the cytoplasm, and suggest that this region may be a modulatory domain that relays growth control signals.

Abstract

Pbx1 is the product of a proto-oncogene originally discovered at the site of chromosomal translocations in acute leukemias. It binds DNA as a complex with a broad subset of homeodomain proteins, but its contributions to hematopoiesis have not been established. This paper reports that Pbx1 is expressed in hematopoietic progenitors during murine embryonic development and that its absence results in severe anemia and embryonic lethality at embryonic day 15 (E15) or E16. Definitive myeloerythroid lineages are present in Pbx1(-/-) fetal livers, but the total numbers of colony-forming cells are substantially reduced. Fetal liver hypoplasia reflects quantitative as well as qualitative defects in the most primitive multilineage progenitors and their lineage-restricted progeny. Hematopoietic stem cells from Pbx1(-/-) embryos have reduced colony-forming activity and are unable to establish multilineage hematopoiesis in competitive reconstitution experiments. Common myeloid progenitors (CMPs), the earliest known myeloerythroid-restricted progenitors, are markedly depleted in Pbx1(-/-) embryos at E14 and display clonogenic defects in erythroid colony formation. Comparative cell-cycle indexes suggest that these defects result largely from insufficient proliferation. Megakaryocyte- and erythrocyte-committed progenitors are also reduced in number and show decreased erythroid colony-forming potential. Taken together, these data indicate that Pbx1 is essential for the function of hematopoietic progenitors with erythropoietic potential and that its loss creates a proliferative constriction at the level of the CMP. Thus, Pbx1 is required for the maintenance, but not the initiation, of definitive hematopoiesis and contributes to the mitotic amplifications of progenitor subsets through which mature erythrocytes are generated. (Blood. 2001;98:618-626)

Abstract

Mammalian Pbx genes (Pbx1-3) encode a family of TALE homeodomain proteins that function as transcriptional regulators in numerous cell types (Curr. Opin. Genet. Dev. 8 (1998) 423). The present study highlights distinctive features of Pbx1b expression during mouse embryonic development as a framework to understand its biological functions. Immunohistochemical analyses demonstrate extensive expression of Pbx1b throughout post-implantation development, with highest levels observed during early to mid-gestation. Its initial distribution is predominantly associated with condensing mesoderm, however, Pbx1b displays dynamic expression patterns in derivatives of all principal germ layers. In particular, Pbx1b localizes to sites of mesenchymal-epithelial interactions during periods of active morphogenesis in tissues such as the lung, kidney, tooth buds and vibrissae follicles. Furthermore, BrdU labeling studies reveal that Pbx1b expression domains partially overlap with regions of cellular proliferation. Taken together, these data suggest that Pbx1b contributes to multiple cellular processes during embryogenesis, which may include roles in cell-autonomous regulation as well as in the mediation of tissue interactions.

Abstract

The t(11;19)(q23;p13.1) chromosomal translocation in acute myeloid leukemias fuses the gene encoding transcriptional elongation factor ELL to the MLL gene with consequent expression of an MLL-ELL chimeric protein. To identify potential mechanisms of leukemogenesis by MLL-ELL, its transcriptional and oncogenic properties were investigated. Fusion with MLL preserves the transcriptional elongation activity of ELL but relocalizes it from a diffuse nuclear distribution to the nuclear bodies characteristic of MLL. Using a serial replating assay, it was demonstrated that the MLL-ELL chimeric protein is capable of immortalizing clonogenic myeloid progenitors in vitro after its retroviral transduction into primary murine hematopoietic cells. However, a structure-function analysis indicates that the elongation domain is not essential for myeloid transformation because mutants lacking elongation activity retain a potent ability to immortalize myeloid progenitors. Rather, the highly conserved carboxyl terminal R4 domain is both a necessary and a sufficient contribution from ELL for the immortalizing activity associated with MLL-ELL. The R4 domain demonstrates potent transcriptional activation properties and is required for transactivation of a HoxA7 promoter by MLL-ELL in a transient transcriptional assay. These data indicate that neoplastic transformation by the MLL-ELL fusion protein is likely to result from aberrant transcriptional activation of MLL target genes. Thus, in spite of the extensive diversity of MLL fusion partners, these data, in conjunction with previous studies of MLL-ENL, suggest that conversion of MLL to a constitutive transcriptional activator may be a general model for its oncogenic conversion in myeloid leukemias. (Blood. 2000;96:3887-3893)

Abstract

PBX1 is a proto-oncogene that plays important roles in pattern formation during development. It was discovered as a fusion with the E2A gene after chromosomal translocations in a subset of acute leukemias. The resulting E2a-Pbx1 chimeric proteins display potent oncogenic properties that appear to require dimerization with Hox DNA binding partners. To define molecular pathways that may be impacted by E2a-Pbx1, a genetic screen consisting of neonatal retroviral infection was used to identify genes that accelerate development of T-cell tumors in E2A-PBX1 transgenic mice. Retroviral insertions in the Notch1 gene were observed in 88% of tumors arising with a shortened latency. Among these, approximately half created a Notch(IC) allele, encoding the intracellular, signaling portion of Notch1, suggesting a synergistic interaction between the Notch and E2a-Pbx1 pathways in oncogenesis. The remaining proviral insertions involving Notch1 occurred in a more 3' exon, resulting in truncating mutations that deleted the carboxy-terminal region of Notch1 containing negative regulatory sequences (Notch1(DeltaC)). In contrast to Notch(IC), forced expression of Notch1(DeltaC) in transgenic mice did not perturb thymocyte growth or differentiation. However, mice transgenic for both the E2A-PBX1 and Notch1(DeltaC) genes displayed a substantially shortened latency for tumor development compared with E2A-PBX1 single transgenic mice. These studies reveal a novel mechanism for oncogenic activation of Notch1 and demonstrate a collaborative relationship between 2 cellular oncogenes that also contribute to cell fate determination during embryonic development. (Blood. 2000;96:1906-1913)

Abstract

Mammalian SET domain-containing proteins define a distinctive class of chromatin-associated factors that are targets for growth control signals and oncogenic activation. SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9, contains both SET and chromo domains, signature motifs for proteins that contribute to epigenetic control of gene expression through effects on the regional organization of chromatin structure. In this report we demonstrate that SUV39H1 represses transcription in a transient transcriptional assay when tethered to DNA through the GAL4 DNA binding domain. Under these conditions, SUV39H1 displays features of a long-range repressor capable of acting over several kilobases to silence basal promoters. A possible role in chromatin-mediated gene silencing is supported by the localization of exogenously expressed SUV39H1 to nuclear bodies with morphologic features suggestive of heterochromatin in interphase cells. In addition, we show that SUV39H1 is phosphorylated specifically at the G(1)/S cell cycle transition and when forcibly expressed suppresses cell growth. Growth suppression as well as the ability of SUV39H1 to form nuclear bodies and silence transcription are antagonized by the oncogenic antiphosphatase Sbf1 that when hyperexpressed interacts with the SET domain and stabilizes the phosphorylated form of SUV39H1. These studies suggest a phosphorylation-dependent mechanism for regulating the chromatin organizing activity of a mammalian su(var) protein and implicate the SET domain as a gatekeeper motif that integrates upstream signaling pathways to epigenetic regulation and growth control.

Abstract

Specific Hox genes are implicated in leukemic transformation, and their selective genetic collaboration with TALE homeobox genes, Pbx and Meis, accentuates their oncogenic potential. The molecular mechanisms underlying these coordinate functions, however, have not been characterized. In this study, we demonstrate that HoxA9 requires its Pbx interaction motif as well as its amino terminus to enhance the clonogenic potential of myeloid progenitors in vitro. We further show that HoxA9 forms functional trimeric DNA binding complexes with Pbx and Meis-like proteins on a modified enhancer. DNA binding complexes containing HoxA9 and TALE homeoproteins display cooperative transcriptional activity and are present in leukemic cells. Trimeric complex formation on its own, however, is not sufficient for HoxA9-mediated immortalization. Rather, structure-function analyses demonstrate that domains of HoxA9 which are necessary for cellular transformation are coincident with those required for trimer-mediated transcriptional activation. Furthermore, the amino terminus of HoxA9 provides essential transcriptional effector properties and its requirement for myeloid transformation can be functionally replaced by the VP16 activation domain. These data suggest that biochemical interactions between HoxA9 and TALE homeoproteins mediate cellular transformation in hematopoietic cells, and that their transcriptional activity in higher order DNA binding complexes provides a molecular basis for their collaborative roles in leukemogenesis.

Abstract

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element.

Abstract

The hepatic leukemia factor (HLF) gene codes for a basic region-leucine zipper (bZIP) protein that is disrupted by chromosomal translocations in a subset of pediatric acute lymphoblastic leukemias. HLF undergoes fusions with the E2A gene, resulting in chimeric E2a-Hlf proteins containing the E2a transactivation domains and the Hlf bZIP DNA binding and dimerization motifs. To investigate the in vivo role of this chimeric bZIP protein in oncogenic transformation, its expression was directed to the lymphoid compartments of transgenic mice. Within the thymus, E2a-Hlf induced profound hypoplasia, premature involution, and progressive accumulation of a T-lineage precursor population arrested at an early stage of maturation. In the spleen, mature T cells were present but in reduced numbers, and they lacked expression of the transgene, suggesting further that E2a-Hlf expression was incompatible with T-cell differentiation. In contrast, mature splenic B cells expressed E2a-Hlf but at lower levels and without apparent adverse or beneficial effects on their survival. Approximately 60% of E2A-HLF mice developed lymphoid malignancies with a mean latency of 10 months. Tumors were monoclonal, consistent with a requirement for secondary genetic events, and displayed phenotypes of either mid-thymocytes or, rarely, B-cell progenitors. We conclude that E2a-Hlf disrupts the differentiation of T-lymphoid progenitors in vivo, leading to profound postnatal thymic depletion and rendering B- and T-cell progenitors susceptible to malignant transformation.

Growth stimulation of primary B cell precursors by the anti-phosphatase Sbf1PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICADe Vivo, I., Cui, X. M., Domen, J., Cleary, M. L.1998; 95 (16): 9471-9476

Abstract

SET binding factor 1 (Sbf1) was originally discovered by virtue of its interaction with a highly conserved motif (the SET domain) of unknown function in the protooncoprotein homolog of Drosophila trithorax, Hrx. Sbf1 shares extensive sequence similarity with myotubularin, a dual specificity phosphatase (dsPTPase) that is mutated in a subset of patients with inherited myopathies. Both Sbf1 and myotubularin interact with the SET domains of Hrx and other epigenetic regulatory proteins, but Sbf1 lacks phosphatase activity due to several evolutionarily conserved amino acid changes in its structurally preserved catalytic pocket. Thus, Sbf1 has features of an anti-phosphatase that could competitively antagonize dsPTPases; however the in vivo role for such factors remains unknown. Given its ability to physically interact with Hrx, a developmental regulator subject to translocation-induced mutations in B cell precursor leukemias, the current studies were undertaken to assess the effects of Sbf1 on lymphopoiesis. After infection with recombinant Sbf1 retroviruses, bone marrow cells were plated under Whitlock-Witte conditions for long-term culture of B lineage cells. Sbf1-expressing cells rapidly dominated the cultures resulting in clonal outgrowths of B cell progenitors that retained a dependence on their primary bone marrow-derived stroma for continuous growth in vitro. Structure/function analyses demonstrated that the SET interaction domain of Sbf1 was necessary and sufficient for growth alterations of B cell progenitors. These observations support a model in which Sbf1 functions as a SET domain-dependent positive regulator of growth-inducing kinase signaling pathways that impinge on SET domain proteins. SET domain-dsPTPase interactions appear to be critically important for regulating the growth properties of B cell progenitors.

Abstract

Several proteins that contribute to epigenetic mechanisms of gene regulation contain a characteristic motif of unknown function called the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) domain. We have demonstrated that SET domains mediate highly conserved interactions with a specific family of proteins that display similarity with dual-specificity phosphatases (dsPTPases). These include myotubularin, the gene of which is mutated in a subset of patients with X-linked myotubular myopathy, and Sbf1, a newly isolated homologue of myotubularin. In contrast with myotubularin, Sbf1 lacks a functional catalytic domain which dephosphorylates phospho-tyrosine and serine-containing peptides in vitro. Competitive interference of endogenous SET domain-dsPTPase interactions by forced expression of Sbf1 induced oncogenic transformation of NIH 3T3 fibroblasts and impaired the in vitro differentiation of C2 myoblast cells. We conclude that myotubularin-type phosphatases link SET-domain containing components of the epigenetic regulatory machinery with signalling pathways involved in growth and differentiation.

Abstract

The HRX gene (also called MLL, ALL-1, and Htrx) at chromosome band 11q23 is associated with specific subsets of acute leukemias through translocations that result in its fusion with a variety of heterologous partners. Two of these partners, ENL and AF9, code for proteins that are highly similar to each other and as fusions with HRX induce myeloid leukemias in mice as demonstrated by retroviral gene transfer and knock-in experiments, respectively. In the present study, a structure-function analysis was performed to determine the molecular requirements for in vitro immortalization of murine myeloid cells by HRX-ENL. Deletions of either the AT hook motifs or the methyltransferase homology domain of HRX substantially impaired the transforming effects of HRX-ENL. The methyltransferase homology domain was shown to bind non-sequence specifically to DNA in vitro, providing evidence that the full transforming activity of HRX-ENL requires multiple DNA binding structures in HRX. The carboxy-terminal 84 amino acids of ENL, which encode two predicted helical structures highly conserved in AF9, were necessary and sufficient for transformation when they were fused to HRX. Similarly, mutations that deleted one or both of these conserved helices completely abrogated the transcriptional activation properties of ENL. This finding correlates, for the first time, a biological function of an HRX fusion partner with the transforming activity of the chimeric proteins. Our studies support a model in which HRX-ENL induces myeloid transformation by deregulating subordinate genes through a gain of function contributed by the transcriptional effector properties of ENL.

Abstract

Mice transgenic for the leukemia oncogene E2A-PBX1 invariably develop lethal, high-grade T-cell lymphomas by 5 months of age. In this study, retroviral insertional mutagenesis was employed to identify oncogenes that cooperate with the E2A-PBX1 transgene in lymphomagenesis. Neonatal retroviral infection substantially reduced length of survival due to accelerated development of lymphomas (81 versus 130 days). The Pim1 gene was targeted by retroviral insertions in 48% of accelerated lymphomas whereas less than 5% contained activated c-Myc and none contained activated Pim2. However, Pim1 DNA rearrangements were frequently sub-stoichiometric and not present at all sites of involvement in an otherwise monoclonal lymphoma indicating that Pim1 activation occurred late in the course of lymphomagenesis. Tumor subpopulations containing activated Pim1 alleles displayed a substantial growth advantage over Pim1 negative cells following serial transfer to secondary, syngeneic recipients. Cooperative interactions were observed in intercrossed Pim1 and E2A-PBX1 transgenic mice in which all double transgenic progeny developed lethal, diffuse T lineage lymphomas by 3 months of age, whereas only 13% of E2A-PBX1 and none of Pim1 single transgenic intercross progeny developed lymphomas by 1 year. Tumors from double transgenic mice were monoclonal providing evidence that additional genetic events were required for transformation. Therefore, Pim1 and E2a-Pbx1 cooperate in T lineage lymphomagenesis but they are not sufficient and the role of Pim1 is more likely to be associated with tumor progression.

Abstract

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

Abstract

A subset of chromosomal translocations in acute leukemias results in the fusion of the trithorax-related protein HRX with a variety of heterologous proteins. In particular, leukemias with the t(11;19)(q23;p13.3) translocation express HRX-ENL fusion proteins and display features which suggest the malignant transformation of myeloid and/or lymphoid progenitor(s). To characterize directly the potential transforming effects of HRX-ENL on primitive hematopoietic precursors, the fusion cDNA was transduced by retroviral gene transfer into cell populations enriched in hematopoietic stem cells. The infected cells had a dramatically enhanced potential to generate myeloid colonies with primitive morphology in vitro. Primary colonies could be replated for at least three generations in vitro and established primitive myelomonocytic cell lines upon transfer into suspension cultures supplemented with interleukin-3 and stem cell factor. Immortalized cells contained structurally intact HRX-ENL proviral DNA and expressed a low-level of HRX-ENL mRNA. In contrast, wild-type ENL or a deletion mutant of HRX-ENL lacking the ENL component did not demonstrate in vitro transforming capabilities. Immortalized cells or enriched primary hematopoietic stem cells transduced with HRX-ENL induced myeloid leukemias in syngeneic and SCID recipients. These studies demonstrate a direct role for HRX-ENL in the immortalization and leukemic transformation of a myeloid progenitor and support a gain-of-function mechanism for HRX-ENL-mediated leukemogenesis.

Abstract

The chimeric oncoprotein E2a-Pbx1 results from fusion of the E2A and PBX1 genes following t(1;19) chromosomal translocations in B cell precursor acute leukemias. Experimentally B cell progenitors do not tolerate constitutive expression of E2a-Pbx1 which contrasts with transformation of several other cell types following its stable expression both in vitro and in vivo. To further investigate the effects of E2a-Pbx1 on the B cell progenitors, we conditionally expressed E2a-Pbx1 under control of a metal response element in hematopoietic precursor cell lines in vitro. Inducible expression of E2a-Pbx1 resulted in cell death with the morphologic and molecular features of apoptosis. A structure-function analysis demonstrated that induction of apoptosis was not a dominant-negative effect of the E2a moiety but, rather, required the DNA-binding homeodomain of Pbx1. E2a-Pbx1-induced apoptosis proceeded through a BCL2-responsive checkpoint eventuating in PARP inactivation but did require p53. Constitutive expression of E2a-Pbx1 did not induce apoptosis or continued cycling of Rat-1 fibroblasts in low serum conditions. These studies demonstrate that E2a-Pbx1 initiates programmed cell death of hematopoietic precursers by a mechanism that requires its chimeric transcriptional properties, but, unlike other nuclear oncoproteins, is independent of p53.

Abstract

E2a-Pbx1 chimeric oncoproteins result from fusion of the E2A and PBX1 genes at the sites of t(1;19) chromosomal translocations in a subset acute lymphoblastic leukemias. Experimentally, E2a-Pbx1 transforms a variety of cell types, including fibroblasts, myeloid progenitors, and lymphoblasts. Structure-function studies have shown that contributions from both E2a and Pbx1 are necessary for oncogenesis, but the Pbx1 homeodomain is dispensable and the required portion of Pbx1 has not been delineated. In this study, we used deletional and site-directed mutagenesis to identify portions of Pbx1 necessary for oncogenic and transcriptional activities of E2a-Pbx1. These studies defined a motif (named the Hox cooperativity motif [HCM]) carboxy terminal to the Pbx homeodomain that is required for cooperative DNA binding, cellular transcriptional activity, and the oncogenic potential of E2a-Pbx1. The HCM is highly conserved throughout the Pbx/exd subfamily of divergent homeodomain proteins and functions in DNA-binding assays as a potential contact site for Hox dimerization. E2a-Pbx1 proteins with interstitial deletion or single-point mutations in the HCM could neither activate transcription in cellular assays nor transform NIH 3T3 cells. An E2a-Pbx1 mutant containing 50 amino acids of Pbx1b spanning the HCM but lacking the homeodomain was capable of inducing fibroblast transformation. Thus, the HCM is a necessary and sufficient contribution of Pbx1 for oncogenesis induced by E2a-Pbx1 and accounts for its homeodomain-independent transforming properties. Since subtle alterations of the Pbx HCM result in complete abrogation of transforming activity whereas the homeodomain is entirely dispensable, we conclude that interactions mediated by the HCM are more important for transformation by E2a-Pbx1 than interactions with cognate Pbx DNA sites.

Abstract

Using the yeast two-hybrid system, we have isolated a cDNA (designated BBP, for Bcl2-binding protein) for a protein (Bbp) that interacts with Bcl2. Bbp is identical to 53BP2, a partial clone of which was previously isolated in a two-hybrid screen for proteins that interact with p53. In this study, we show that specific interactions of Bbp/53BP2 with either Bcl2 or p53 require its ankyrin repeats and SH3 domain. These interactions can be reproduced in vitro with bacterially expressed fusion proteins, and competition experiments indicate that Bcl2 prevents p53 from binding to Bbp/53BP2. BBP/53BP2 mRNA is abundant in most cell lines examined, but the protein cannot be stably expressed in a variety of cell types by transfection. In transiently transfected cells, Bbp partially colocalizes with Bcl2 in the cytoplasm and results in an increased number of cells at G2/M, possibly accounting for the inability to obtain stable transfectants expressing the protein. These results demonstrate that a single protein can interact with either Bcl2 or p53 both in yeast cells and in vitro. The in vivo significance of these interactions and their potential consequences for cell cycle progression and cell death remain to be determined.

Abstract

True histiocytic lymphomas (THLs) are rare tumors in which the malignant cells show morphologic and immunophenotypic evidence of histiocytic differentiation. We describe THLs that arose after therapy for one case of T-lineage lymphoblastic lymphoma (LyL) and two cases of acute lymphoblastic leukemia (ALL) (both CD10+, one pre-B phenotype). The lymphoblastic neoplasms were not unusual in any way, and responded well to standard therapy. The THLs arose 10 to 20 months after complete remission was achieved for the lymphoblastic neoplasms, at which time there was still no clinical or pathologic evidence of the lymphoblastic neoplasms. All three THLs exhibited clinical and morphologic features of malignancy. Neoplastic cells in the THLs had abundant eosinophilic vacuolated cytoplasm and pleomorphic nuclei, and expressed histiocytic antigens in the absence of lymphocyte-specific lineage markers. Because THLs are rare neoplasms, their occurrence after otherwise successful therapy for lymphoblastic neoplasms in these three cases may constitute a distinct clinicopathologic entity.

Abstract

LYL1 is a basic helix-loop-helix (HLH) protein that was originally discovered because of its translocation into the beta T-cell receptor locus in an acute lymphoblastic leukemia. LYL1 is expressed in many hematolymphoid cells, with the notable exceptions of thymocytes and T cells. Using the yeast two-hybrid system to screen a cDNA library constructed from B cells, we identified the E-box-binding proteins E12 and E47 as potential lymphoid dimerization partners for LYL1. The interaction of LYL1 with E2a proteins was further characterized in vitro and shown to require the HLH motifs of both proteins. Immunoprecipitation analyses showed that in T-ALL and other cell lines, endogenous LYL1 exists in a complex with E2a proteins. A preferred DNA-binding sequence, 5'-AACAGATG(T/g)T-3', for the LYL1-E2a heterodimer was determined by PCR-assisted site selection. Endogenous protein complexes containing both LYL1 and E2a bound this sequence in various LYL1-expressing cell lines and could distinguish between the LYL1 consensus and muE2 sites. These data demonstrate that E2a proteins serve as dimerization partners for the basic HLH protein LYL1 to form complexes with distinctive DNA-binding properties and support the hypothesis that the leukemic properties of the LYL1 and TAL subfamily of HLH proteins could be mediated by recognition of a common set of target genes as heterodimeric complexes with class I HLH proteins.

Abstract

Pbx cofactors are implicated to play important roles in modulating the DNA-binding properties of heterologous homeodomain proteins, including class I Hox proteins. To assess how Pbx proteins influence Hox DNA-binding specificity, we used a binding-site selection approach to determine high-affinity target sites recognized by various Pbx-Hox homeoprotein complexes. Pbx-Hox heterodimers preferred to bind a bipartite sequence 5'-ATGATTNATNN-3' consisting of two adjacent half sites in which the Pbx component of the heterodimer contacted the 5' half (ATGAT) and the Hox component contacted the more variable 3' half (TNATNN). Binding sites matching the consensus were also obtained for Pbx1 complexed with HoxA10, which lacks a hexapeptide but requires a conserved tryptophan-containing motif for cooperativity with Pbx. Interactions with Pbx were found to play an essential role in modulating Hox homeodomain amino-terminal arm contact with DNA in the core of the Hox half site such that heterodimers of different compositions could distinguish single nucleotide alterations in the Hox half site both in vitro and in cellular assays measuring transactivation. When complexed with Pbx, Hox proteins B1 through B9 and A10 showed stepwise differences in their preferences for nucleotides in the Hox half site core (TTAT to TGAT, 5' to 3') that correlated with the locations of their respective genes in the Hox cluster. These observations demonstrate previously undetected DNA-binding specificity for the amino-terminal arm of the Hox homeodomain and suggest that different binding activities of Pbx-Hox complexes are at least part of the position-specific activities of the Hox genes.

Abstract

True histiocytic lymphoma (THL), as it is currently defined, is a rare entity. We report 12 cases of THL seen at Stanford over the last ten years. By definition, the neoplastic cells in each case showed histological and immunological evidence of histiocytic differentiation. Seven females and five males ranged in age from 9 to 67 years. Sites of involvement included lymph node, soft tissue, bone, stomach, small intestine, mediastinum, kidney, breast and salivary gland. Lymph nodes showed diffuse architectural effacement and/or a paracortical pattern of involvement. The infiltrates involved other tissues in a diffuse pattern. Cytologically the cells were characterized by abundant eosinophilic cytoplasm and enlarged, indented eccentrically placed nuclei containing prominent nucleoli. In all cases the cytological features were sufficiently atypical to indicate a neoplastic infiltrate. Paraffin section immunophenotyping demonstrated reactivity of the atypical cells for CD15, 43, 45RO, 45RB, 68, lysozyme and/or S100. In frozen sections, the atypical cells demonstrated reactivity for CD4 (cytoplasmic), 11c, 14, 15, and/or 68. Genotypic studies were performed on 3 cases, one of which showed rearrangements of immunoglobulin heavy and light chain genes. Follow-up was available on eleven patients, six of whom died of disease 0.5 to 36 months following diagnosis.

Abstract

The human proto-oncogene PBX1 codes for a homolog of Drosophila extradenticle, a divergent homeo domain protein that modulates the developmental and DNA-binding specificity of select HOM proteins. We demonstrate that wild-type Pbx proteins and chimeric E2a-Pbx1 oncoproteins cooperatively bind a consensus DNA probe with HoxB4, B6, and B7 of the Antennapedia class of Hox/HOM proteins. Specificity of Hox-Pbx interactions was suggested by the inability of Pbx proteins to cooperatively bind the synthetic DNA target with HoxA10 or Drosophila even-skipped. Site-directed mutagenesis showed that the hexapeptide motif (IYPWMK) upstream of the Hox homeo domain was essential for HoxB6 and B7 to cooperatively bind DNA with Pbx proteins. Engraftment of the HoxB7 hexapeptide onto HoxA10 endowed it with robust cooperative properties, demonstrating a functional role for the highly conserved hexapeptide element as one of the molecular determinants delimiting Hox-Pbx cooperativity. The Pbx homeo domain was necessary but not sufficient for cooperativity, which required conserved amino acids carboxy-terminal of the homeo domain. These findings demonstrate that interactions between Hox and Pbx proteins modulate their DNA-binding properties, suggesting that Pbx and Hox proteins act in parallel as heterotypic complexes to regulate expression of specific subordinate genes.

Abstract

Virus-associated hemophagocytic syndromes are a heterogeneous group of disorders in which viral infection is associated with a proliferation of hemophagocytic histiocytes throughout the reticuloendothelial system. The authors report the case of a 24-year-old Vietnamese male who developed a hemophagocytic syndrome associated with Epstein-Barr virus (EBV) and who died following a rapidly progressive course. A proliferation of reactive-appearing lymphoid cells was associated with an extensive proliferation of erythrophagocytic histiocytes. Immunophenotypically, the lymphoid infiltrate consisted of CD56+ natural killer cells, predominantly CD8+ T-cells and rare B-cells (CD20+). Double-label immunohistochemical studies showed CD3+ T-cells and CD56+ natural killer cells to be distinct cell populations. Combined immunohistochemical-in situ hybridization studies localized EBV to CD43+, CD3-, CD68-, lymphoid-appearing cells, indicating the presence of EBV within natural killer cells. Southern hybridization analysis of EBV genomic termini revealed clonal EBV genome. However, there was no detectable immunoglobulin or T-cell receptor gene rearrangements. The findings indicate that this case of hemophagocytic syndrome represents a clonal proliferation of natural killer cells containing EBV and highlights the importance of the analysis of EBV genomic termini for determination of clonality in EBV-associated proliferations. It is possible that other cases of fulminant EBV-associated hemophagocytic syndromes represent clonal natural killer cell proliferations.

Abstract

The t(1;19) chromosomal translocation in acute lymphoblastic leukemias creates chimeric E2a-Pbx1 oncoproteins that can act as DNA-binding activators of transcription. A structural analysis of the functional domains of E2a-Pbx1 showed that portions of both E2a and Pbx1 were essential for transformation of NIH 3T3 cells and transcriptional activation of synthetic reporter genes containing PBX1 consensus binding sites. Hyperexpression of wild-type or experimentally truncated Pbx1 proteins was insufficient for transformation, consistent with their inability to activate transcription. When fused with E2a, the Pbx-related proteins Pbx2 and Pbx3 were also transformation competent, demonstrating that all known members of this highly similar subfamily of homeodomain proteins have latent oncogenic potential. The oncogenic contributions of E2a to the chimeras were localized to transactivation motifs AD1 and AD2, as their mutation significantly impaired transformation. Either the homeodomain or Pbx1 amino acids flanking this region could mediate transformation when fused to E2a. However, the homeodomain was not essential for transformation, since a mutant E2a-Pbx1 protein (E2a-Pbx delta HD) lacking the homeodomain efficiently transformed fibroblasts and induced malignant lymphomas in transgenic mice. Thus, transformation mediated by the chimeric oncoprotein E2a-Pbx1 is absolutely dependent on motifs acquired from E2a but the Pbx1 homeodomain is optional. The latter finding suggests that E2a-Pbx1 may interact with cellular proteins that assist or mediate alterations in gene expression responsible for oncogenesis even in the absence of homeodomain-DNA interactions.

Abstract

Chromosome band 11q23 is the site of recurring translocations with a variety of partner chromosomes in myeloid and lymphoid acute leukemias, infant leukemias, and treatment-induced secondary acute myelogenous leukemia. The translocation breakpoints cluster in a restricted region of the HRX gene resulting in fusion genes that encode an N-terminal portion of Hrx fused to various partner proteins. We have characterized the transcriptional transactivation properties of Enl, a protein that is fused to Hrx in t(11;19) leukemias. Enl is a nuclear protein that is capable of activating transcription from synthetic reporter genes in both lymphoid and myeloid cells, as well as in yeast. Deletion mutagenesis demonstrated that the minimal portion of Enl required for activation of transcription was localized to its C-terminal 90 amino acids. This region is highly conserved between Enl and the t(9;11) fusion partner Af-9 and is retained in all Hrx-Enl and Hrx-Af9 fusion proteins. Thus, the leukemogenic contribution and transcriptional activation potential of Enl colocalize to its highly conserved carboxy terminus, suggesting that Hrx-Enl chimeric proteins mediate alterations in the transcription program of t(11;19)-bearing cells.

Abstract

We describe the first case of an Epstein-Barr virus (EBV)-associated natural killer-large granular lymphocyte (NK-LGL) leukemia in the United States to the best of our knowledge. A 29-year-old woman of Japanese descent developed EBV infection after a blood transfusion as indicated by a rise in serum antibody titers. Peripheral blood and bone marrow aspirate smears demonstrated increased LGLs. Flow cytometry showed that these cells expressed NK-associated surface antigens. Cytogenetic analysis of the bone marrow aspirate showed two distinct but related clones with multiple copies of a modified 7 marker chromosome. Death followed colonic perforation. Findings at necropsy included bone marrow lymphocytosis and erythrophagocytosis, a mononucleosis-like lymphadenitis, atypical hepatitis with a mixed, predominantly T-cell infiltrate, interstitial pneumonitis, and multiorgan system vasculitis with perforation of the transverse colon. Epstein-Barr virus transcripts were identified in lymphocytes infiltrating liver and peripheral nerve by in situ hybridization. In addition, Southern blot analyses showed monoclonal bands superimposed on oligoclonal ladders of EBV termini in liver and lymph node. The identical episomal form of EBV was found in the bone marrow, lymph node, and liver. No immunoglobulin (Ig), T-cell receptor beta, or T-cell receptor gamma chain gene rearrangements were identified. These studies support the hypothesis that the LGL population was a neoplastic EBV-related clonal proliferation of NK cells.

Abstract

The t(17;19) translocation in acute lymphoblastic leukemias results in creation of E2A-hepatic leukemia factor (HLF) chimeric proteins that contain the DNA-binding and protein dimerization domains of the basic leucine zipper (bZIP) protein HLF fused to a portion of E2A proteins with transcriptional activation properties. An in vitro binding site selection procedure was used to determine DNA sequences preferentially bound by wild-type HLF and chimeric E2A-HLF proteins isolated from various t(17;19)-bearing leukemias. All were found to selectively bind the consensus sequence 5'-GTTACGTAAT-3' with high affinity. Wild-type and chimeric HLF proteins also bound closely related sites identified previously for bZIP proteins of both the proline- and acidic amino acid-rich (PAR) and C/EBP subfamilies; however, E2A-HLF proteins were significantly less tolerant of certain deviations from the HLF consensus binding site. These differences were directly attributable to loss of an HLF ancillary DNA-binding domain in all E2A-HLF chimeras and were further exacerbated by a zipper mutation in one isolate. Both wild-type and chimeric HLF proteins displayed transcriptional activator properties in lymphoid and nonlymphoid cells on reporter genes containing HLF or C/EBP consensus binding sites. But on reporter genes with nonoptimal binding sites, their transcriptional properties diverged and E2A-HLF competitively inhibited activation by wild-type PAR proteins. These findings establish a spectrum of binding site-specific transcriptional properties for E2A-HLF which may preferentially activate expression of select subordinate genes as a homodimer and potentially antagonize expression of others through heteromeric interactions.

Abstract

Chromosome band 11q23, the location of the HRX gene, is a site of recurrent translocations in human malignancies. Infants with acute lymphoblastic leukemia (ALL) commonly have 11q23 translocations and have an especially poor prognosis despite intensive chemotherapy. We analyzed 96 cases of infant ALL treated on three consecutive Pediatric Oncology Group protocols to determine the frequency and prognostic significance of molecular rearrangements of HRX. Overall, 78 cases (81%) had HRX rearrangements detected by Southern blot analysis performed with a single HRX cDNA probe, whereas 18 cases (19%) had germline HRX. Of the 78 cases with HRX rearrangements, only 50 had abnormalities of 11q23 detected cytogenetically. Molecular abnormalities of HRX were associated with early treatment failure and a very poor outcome. Estimated event-free survival for patients with HRX rearrangements was 19% (SE, 7%) at 3 years, compared with 46% (SE, 17%) for patients with germline HRX (P = .033 by the two-sided logrank test). Therefore, infants with ALL and molecular abnormalities of HRX represent a group with an extremely high rate of failure who clearly need innovative or experimental treatment. Furthermore, cytogenetic analysis alone failed to detected 36% of HRX rearrangements, suggesting that molecular analysis be performed on all infants with ALL to identify this group of high-risk patients.

Abstract

The t(1;19) chromosomal translocation is observed in pre-B cell acute lymphoblastic leukemias and results in expression of chimeric E2A-PBX1 proteins that contain transcriptional activation domains from E2A and the homeodomain of PBX1. Since homeodomains mediate DNA-binding, a potential model for the action of E2A-PBX1 is that it disrupts the transcriptional regulation of genes normally controlled by PBX1 or its closely-related family members PBX2 or PBX3. Using a binding site selection assay, we identified a consensus nucleotide sequence ATCAATCA specifically bound by the PBX1 homeodomain and those of its closely-related family members PBX2 and PBX3. An endogenous protein with the properties of PBX3b specifically bound to this sequence in nuclear extracts of precursor B cells. Transfection of reporter genes containing PBX binding sites linked to a minimal promoter demonstrated transactivation by E2A-PBX1 fusion protein dependent upon presence of the homeodomain. In contrast, wild-type PBX proteins were incapable of activating transcription. The striking differences in transcriptional properties of fusion and wild-type PBX proteins provides strong functional evidence for the importance of aberrant transcriptional regulation in the genesis of t(1;19)-bearing leukemias.

Abstract

The t(17;19)(q21-q22;p13.3) in acute lymphoblastic leukemia results in creation of an E2A-HLF fusion protein with structural and functional properties of a chimeric transcription factor. Two types of genomic rearrangements underlie fusion of E2A with HLF. In type I rearrangements, an insertion that codes for a portion of the chimera not found in either wild type protein occurs between E2A exon 13- and HLF exon 4-encoded sequences. This insertion is derived from a cryptic exon created at the junction between chromosomes 17 and 19, and includes intronic portions of both E2A and HLF with intervening nontemplated N nucleotides. Type II rearrangements arise from more 5' breakpoints in E2A and result in fusion cDNAs with E2A exon 12 spliced directly to HLF exon 4. Analysis of the genomic structure of HLF shows that these different modes of protein fusion result from selective constraints to maintain the proper HLF reading frame, because a direct E2A exon 13 to HLF exon 4 splice would lead to translation of a truncated E2A protein lacking any contribution from HLF. These features underscore the requirement for DNA binding and/or dimerization conferred by the bZIP portion of the E2A-HLF chimera in t(17;19)-ALL.

Abstract

The Bcl2 protein inhibits apoptosis (programmed cell death) induced by a variety of noxious stimuli. However, relatively little is known about its effect on apoptosis that occurs after terminal differentiation. Bcl2 protein levels decrease during differentiation of myeloid cells into granulocytes that subsequently undergo apoptosis, but the potential role of Bcl2 in coupling survival and differentiation remains undefined. To ascertain the relationship between decreasing Bcl2 levels and the onset of apoptosis in differentiating myeloid cells, Bcl2 was hyperexpressed in the HL-60 cell line after retroviral gene transfer. After treatment of HL-60/BCL2 cells with all-trans retinoic acid or phorbol myristic acid, Bcl2 levels did not decrease as in normal HL-60 cells but, rather, increased because of activation of the viral promoter. Differentiation of the Bcl2-overexpressing cells was similar to that of normal HL-60 cells, but they showed little evidence for apoptosis and had a prolonged survival. These studies show that the survival-enhancing properties of Bcl2 counteract programmed cell death that accompanies terminal differentiation; however, Bcl2 has no significant effect on differentiation itself, suggesting that apoptosis and differentiation are regulated independently in myeloid cells.

Abstract

Expression of the bcl-2 proto-oncogene on chromosome 18 is deregulated by the 14; 18 chromosomal translocation, an abnormality that is consistently associated with follicular non-Hodgkin's lymphomas (NHL). Because bcl-2 is believed to function by prolonging cell survival rather than by increasing proliferation, the presence of t(14; 18) in Hodgkin's disease (HD) would have profound implications for the pathogenesis of this neoplasm. We evaluated 32 cases of HD for t(14; 18) by polymerase chain reaction (PCR). These results were correlated with expression of bcl-2 oncogenic protein by Hodgkin cells and with the presence of Epstein-Barr virus (EBV), as determined by immunohistochemistry or in situ hybridization. PCR provided evidence of t(14; 18) in only 2 HD cases (6%), both of which were associated with a prior history of follicular lymphoma, and both of which were among the 7 cases (22%) with strong bcl-2 expression in Hodgkin cells. In at least 1 of the cases, the translocation involved identical chromosomal breakpoints in both types of lymphoma. Furthermore, 7 additional cases of combined follicular NHL and HD showed strong bcl-2 staining in Hodgkin cells. Although EBV was detected in 6 of 30 cases, it was not associated with t(14; 18) and usually not with strong bcl-2 expression. These results suggest that a small proportion of HD cases might evolve from follicular NHL, possibly through molecular events superimposed on the t(14; 18). High-level bcl-2 expression in Hodgkin cells is a potentially useful but not definitive marker for these cases.

Abstract

A major mode of proto-oncogene activation by chromosomal translocations concerns the creation of fusion genes which encode chimaeric proteins. The largest class of oncogenes identified to date is the transcription factors, which are involved in control of cellular proliferation and differentiation via regulation of target gene transcription. Protein chimaeras that result from translocations in childhood ALL include two which involve the 19p13.3 gene E2A and a large heterogeneous group involving HRX located at chromosome band 11q23. Functional studies demonstrate that E2A fusion proteins function as chimaeric transcription factors, and structural features suggest that HRX fusion proteins may have analogous properties.

Abstract

Expression of the homeobox fusion gene E2A-PBX1 under control of the immunoglobulin heavy chain enhancer efficiently induced malignancies in transgenic mice. All animals died before 5 months of age with lymphomas that demonstrated phenotypes consistent with transitional intermediate thymocytes (CD4+/CD8+/CD3med). E2A-PBX1 also markedly altered lymphoid development in pretumorous animals, reducing the number of thymocytes and bone marrow B lineage progenitors to 20% of normal levels. In spite of the observed reductions in lymphoid cells, premalignant animals contained significantly increased numbers of cycling thymocytes, but a higher proportion was also undergoing apoptosis, suggesting that increased cell death resulted in the marked lymphopenias. These data indicate that the chimeric homeodomain protein E2A-PBX1 paradoxically induces both proliferation and apoptosis in lymphoid cells, suggesting an in vivo association between nuclear oncogene-induced cell cycle progression and programed cell death.

Abstract

Immunosuppressed individuals are at high risk for the development of hematologic malignancies. The typical lymphomas arising in organ transplant recipients are B-cell non-Hodgkin's lymphomas that contain Epstein-Barr virus (EBV) DNA sequences. We investigated the characteristics of posttransplant lymphomas that lacked expression of the usual markers associated with EBV transformation. We describe four large-cell lymphomas seen recently at our institution. Two of these four cases were CD4+, one was CD8+, and in one staining for CD4 and CD8 expression was not performed. One CD4+ lymphoma was a CD30+, EBV- large-cell lymphoma from a 65-year-old kidney transplant recipient, the second was an EBV+ large-cell lymphoma from a 25-year-old heart transplant patient. Two T-cell lymphomas were EBV+ and had clonal T-cell receptor beta gene rearrangements. The other two lymphomas expressed T-cell markers CD4 and CD43, and lacked expression of B-cell markers CD19, CD20, CD21, CD22, CD23, and surface Ig. Both CD4+ lymphomas were tumorigenic after their heterotransplantation into severe combined immunodeficient (SCID) mice. Cytogenetics, immunophenotyping, and genotyping of the secondary tumors from SCID mice showed their clonality and identity with the patients' primary tumors. Novel CD4+ lymphoma cell lines, LH521/4 and LK418/4, were established from tumors that had been passaged in SCID mice. An immunodeficient environment may facilitate the growth of these T-cell or biphenotypic lymphomas; the etiology of their genesis can include transformation with EBV and other, as yet unidentified mechanisms.

Abstract

Chromosome band 11q23 is a site of recurrent translocations and interstitial deletions in human leukemias. Recent studies have shown that the 11q23 gene HRX is fused to heterologous genes from chromosomes 4 or 19 after t(4;11)(q21;q23) and t(11;19)(q23;p13) translocations to create fusion genes encoding proteins with structural features of chimeric transcription factors. In this report, we show structural alterations of HRX by conventional Southern blot analyses in 26 of 27 de novo leukemias with cytogenetically diverse 11q23 abnormalities. The sole case that lacked HRX rearrangements was a t(11;17)-acute myeloid leukemia with French-American-British M3-like morphology. We also analyzed 10 secondary leukemias that arose after therapy with topoisomerase II inhibitors and found HRX rearrangements in 7 of 7 with 11q23 translocations, and in 2 of 2 with unsuccessful karyotypes. In total, we observed HRX rearrangements in 35 leukemias involving at least nine distinct donor loci (1q32, 4q21, 6q27, 7p15, 9p21-24, 15q15, 16p13, and two 19p13 sites). All breakpoints localized to an 8-kb region that encompassed exons 5-11 of HRX, suggesting that fusion proteins containing similar portions of HRX may be consistently created in leukemias with 11q23 abnormalities. We conclude that alteration of HRX is a recurrent pathogenetic event in leukemias with 11q23 aberrations involving many potential partners in a variety of settings including acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia in blast crisis, and topoisomerase II inhibitor-induced secondary leukemias of both the myeloid and lymphoid lineages.

Abstract

Translocations involving chromosome band 11q23 in acute leukemias have recently been shown to involve the HRX gene that codes for a protein with significant similarity to Drosophila trithorax. HRX gene alterations are consistently observed in t(4;11) (q21;q23)-carrying leukemias and cell lines by Southern blot analyses and are accompanied by HRX transcripts of anomalous size on Northern blots. HRX-homologous cDNAs were isolated from a library prepared from t(4;11)-carrying acute leukemia cells. cDNAs representative of transcription products from the derivative 11 chromosome were shown to contain HRX sequences fused to sequences derived from chromosome band 4q21. Fragments of the latter were used to clone and analyze cDNAs for wild-type 4q21 transcripts that predicted a 140-Kd basic protein (named FEL) that is rich in prolines, serines, and charged amino acids. FEL contains guanosine triphosphate-binding and nuclear localization consensus sequences and uses one of two possible 5' exons encoding the first 12 or 5 amino acids. After t(4;11) translocations, 913 C-terminal amino acids of FEL are fused in frame to the N-terminal portion of HRX containing its minor groove DNA binding motifs. These features are similar to predicted t(11;19) fusion proteins, suggesting that HRX consistently contributes a novel DNA-binding motif to at least two different chimeric proteins in acute leukemias.

Abstract

We have studied the distribution of the bcl-2 protein in fetal tissues, in an effort to uncover patterns of expression that may elucidate the potential role of bcl-2 during development. We find that bcl-2 is expressed in many hematolymphoid and non-hematolymphoid tissues, most abundantly in placental trophoblast. In tissues of endocrine and neural derivation and in stem-cell populations of colonic and some stratified epithelia, bcl-2 seems to be involved in tissue homeostasis. However, in developing proximal nephrons of the kidney and other sites characterized by inductive interactions between epithelium and mesenchyme, bcl-2 is apparently involved in morphogenesis, possibly by mediating the formation of condensations of cells that are "committed" to the formation of more differentiated structures. The distribution of bcl-2-protein expression in fetal tissues is consistent with its previously described role in promoting cell survival, presumably by preventing apoptosis in lymphoid and other tissues where cell death represents an active regulatory process. Expression of bcl-2 protein is more widespread in fetal than adult tissues. Our observations therefore represent supportive evidence for the importance of inducible cell survival as a regulatory process in normal homeostasis and morphogenesis in many fetal tissues and structures.

Abstract

Immunohistochemistry and in situ hybridization with a synthetic oligonucleotide probe were used to compare the topographical distribution of BCL-2 proto-oncogenic protein with that of its messenger RNA (mRNA) in normal lymphoid tissues, follicular lymphomas, and lymphoma-derived cell lines. In normal lymph nodes, BCL-2 protein was most abundant in the small lymphocytes of primary lymphoid follicles and the mantle zones of secondary follicles, virtually absent within germinal centers, and of variable abundance in many interfollicular cells. In contrast, the distribution of BCL-2 mRNA was roughly reciprocal to that of the protein with intense hybridization signal in germinal centers and almost none in mantle zones. Discordant BCL-2 RNA and protein levels were also observed in tonsillar epithelial cells and cortical thymocytes. Concordant and abundant expression of BCL-2 mRNA and protein was detected in biopsy tissues and cell lines from t(14;18)-carrying lymphomas. The contrasting distributions of BCL-2 protein and RNA in normal lymphoid tissues suggest that translational and posttranslational control mechanisms play a significant role in regulating BCL-2 protein levels in germinal center cells, epithelial cells, and cortical thymocytes. Concordant BCL-2 mRNA and protein levels in follicular lymphomas suggest that translational control mechanisms may be disrupted as part of the sequence of genetic changes that transforms normal lymphoid cells into neoplastic follicular lymphoma cells.

Abstract

We have identified a human homolog of the Drosophila trithorax protein that is structurally altered by 11q23 translocations in acute leukemias. Human trithorax (HRX) is a predicted 431 kd protein containing two potential DNA-binding motifs consisting of zinc fingers conserved with the fly protein and nonconserved amino-terminal "AT hook" motifs related to the DNA-binding motifs in HMG proteins. 11q23 translocations disrupt the HRX gene between these two motifs, and in a t(11;19)-carrying cell line fusion transcripts are expressed from both derivative chromosomes. The more abundant derivative 11 transcript codes for a chimeric protein containing the AT hook motifs fused to a previously undescribed protein (ENL) from chromosome 19. These data suggest a novel role for a trithorax-homologous protein in multilineage human leukemias that may be mediated by DNA binding within the minor groove at AT-rich sites, implicated to play an important role in bacterial IHF-, yeast datin-, and mammalian HMG-mediated gene activation.

Abstract

Oncogenic conversion of transcription factors by chromosomal translocations is implicated in leukemogenesis. We report that the t(17;19) in acute lymphoblastic leukemia produces a chimeric transcription factor consisting of the amino-terminal portion of HLH proteins E12/E47 (products of the E2A gene) fused to the basic DNA-binding and leucine zipper dimerization motifs of a novel hepatic protein called hepatic leukemia factor (Hlf). Hlf, which is not normally transcribed in lymphoid cells, belongs to the recently described PAR subfamily of basic leucine zipper (bZIP) proteins, which also includes Dbp and Tef/Vbp. Wild-type Hlf is able to bind DNA specifically as a homodimer or as a heterodimer with other PAR factors. Structural alterations of the E2a-Hlf fusion protein markedly impair its ability to bind DNA as a homodimer compared with wild-type Hlf. However, E2a-Hlf can bind DNA as a heterodimer with other PAR proteins, suggesting a novel mechanism for leukemogenic conversion of a bZIP transcription factor.

Abstract

The gastrointestinal tract is the most common site for extranodal lymphomas, but follicular lymphomas involving the gut are rare. To study their pathologic features and bcl-2 expression, 31 follicular lymphomas of the GI tract were reviewed and unstained paraffin sections from 24 of the cases were immunohistochemically stained using a monoclonal antibody for the peptide product of the proto-oncogene bcl-2. The most common site of lymphoma involvement was the small intestine, especially the terminal ileum. Gastric lymphomas tended to present clinically with symptomatic ulcers and small intestinal lesions presented with obstruction. Five cases involving the terminal ileum or colon had a gross appearance of multitudinous mucosal polyps and were considered to represent examples of "multiple lymphomatous polyposis." Enhanced expression of the bcl-2 oncogenic protein was detectable in lymphoma cells in 75% of cases and at lower levels in normal lymphoid cells in most cases. Small cleaved or mixed cell lymphomas were more likely to show enhanced expression than were large cell cases. Reactive germinal centers showed no bcl-2 staining. It is concluded that follicular GI lymphomas are associated with distinctive pathological features. In their tendency to express bcl-2, these neoplasms resemble their lymph node-based counterparts. Immunohistochemical staining for enhanced bcl-2 expression is of potential diagnostic utility in distinguishing between follicular lymphoma and follicular lymphoid hyperplasia in the gastrointestinal tract. The relevance of the results to lymphoma of mucosa-associated lymphoid tissue (MALT) is discussed.

Abstract

At the present time, two general mechanisms account for deregulation and subsequent oncogenic conversion of transcriptional proteins in human leukaemias. One involves quantitative alterations in expression, suggesting that activity of the involved factors is primarily controlled by their accessibility within the cell. Neoplastic transformation may result from excessive expression or, conversely, complete loss of functional products (eg tumour suppressor proteins not described here). The second mechanism involves mutation by protein fusion (or truncation) and illustrates the modular composition of transcriptional proteins. The loss or inappropriate combination of specific modules creates chimaeric proteins with presumably altered transcriptional properties that may contribute to the neoplastic phenotype. Both mechanisms underscore the importance of cognate interactions, particularly heterodimerization between various transcriptional proteins with other members of the transcription complex. Future efforts will continue to focus on the interactions of oncogenic transcription factors with other cellular proteins and their biologically relevant target genes.

Abstract

Two new homeobox genes, PBX2 and PBX3, were isolated on the basis of their extensive homology to PBX1, a novel human homeobox gene involved in t(1;19) translocation in acute pre-B-cell leukemias. The predicted Pbx2 and Pbx3 proteins are 92 and 94% identical to Pbx1 over a large region of 266 amino acids within and flanking their homeodomains, but all three proteins diverge significantly near their amino and carboxy termini. Chromosome in situ hybridizations demonstrated that the PBX genes are not clustered but map to separate chromosomal loci: PBX1, 1q23; PBX2, 3q22-23; PBX3, 9q33-34. Expression of PBX2 or PBX3 was not restricted to particular states of differentiation or development, as mRNA transcripts of these genes were detected in most fetal and adult tissues and all cell lines, unlike PBX1, which is not expressed in lymphoid cell lines. Similar to PBX1 RNA, PBX3 RNA is alternatively spliced to yield two translation products with different carboxy termini, a feature not observed for PBX2. Their extensive sequence similarity and widespread expression suggest a generalized, overlapping role for Pbx proteins in most cell types. Differences in their amino and carboxy termini may modulate their activities, mediated in part by differential splicing and, for PBX1, protein fusion following t(1;19) chromosomal translocation.

Abstract

We reasoned that the SCID-hu mouse could provide an appropriate lymphoid or stromal microenvironment to support the growth of primary human lymphoma. Heterotransplantation of nine cases of primary T-cell non-Hodgkin's lymphoma (NHL) into untreated SCID mice and SCID mice reconstituted with human fetal thymus, spleen, and liver (SCID-hu) resulted in the development of lymphoid tumors in five (56%) cases. Two clonal T-cell NHL grew after a mean of 90 days after injection of primary lymphoma cell suspensions into the thymus xenografts in SCID-hu mice and failed to grow in a variety of sites in SCID mice, except for small tumors that developed after a long (157-day) latency period after intracranial injection of tumor cell suspensions into weanling SCID mice. Successful serial transplantation of NHL in SCID and SCID-hu mice required the presence of a human lymphoid or tumor microenvironment, and was enhanced by pretreating the SCID mice with 175 rad radiation and antiasialo antisera. Analysis of the primary and transplanted T-cell tumors showed identical patterns of T-cell surface markers by flow cytometry and immunophenotyping of fixed tissue sections, and, in one case, reactivity with a specific monoclonal antibody to V beta 5.1. Genotyping of the transplanted tumors showed T-cell receptor gene rearrangements identical to those present in the primary tumors. In one case, the presence of Epstein-Barr virus-positive B cells in association with the primary tumor resulted in the growth of a lymphoblastoid B-cell neoplasm in addition to the malignant T-cell lymphoma after transplantation of tumor fragments to SCID mice. The data support the hypothesis that a human lymphoid microenvironment enhances the growth of T-cell NHL in SCID mice. The SCID-hu thymus graft provides an apparently unique microenvironment that supports the growth of primary T-cell NHL, and can be used to study the interaction between lymphoma cells, nontransformed lymphoid cells, and the surrounding stromal microenvironment in vivo.

Abstract

The t(14;18) chromosomal translocation that results in the juxtaposition of the bcl-2 proto-oncogene with the heavy chain JH locus is a common cytogenetic abnormality in human lymphoma. In particular, it is seen in about 85% of follicular lymphoma (FL) and up to one-third of diffuse lymphomas (DL). The chromosome 18 breakpoints have been shown to cluster into two regions. The major breakpoint region (mbr) within the 3' untranslated region of the bcl-2 proto-oncogene accounts for approximately 60% of the cases and the minor cluster region (mcr) 30 kb 3' of bcl-2 accounts for approximately 25% of the breakpoints. Because of variability in the position of the breakpoint, detection of the t(14;18) by Southern blot analysis provides an important clonal marker for the tumor. However, conventional electrophoresis (CE) fails to detect the translocation in 15% to 25% of cases. We have applied pulsed-field gel electrophoresis (PFGE) to the detection of the t(14;18) in a series of lymphoma prospectively analyzed by CE, polymerase chain reaction (PCR), and cytogenetic analysis. PFGE readily detected t(14;18) rearrangements as indicated by comigration of bands detected with probes for the mbr region (chromosome 18) and the JH locus (chromosome 14). In a series of 40 patients with FL, this method proved to be the most comprehensive for detection of the translocation compared with standard methods; in fact, in one case only PFGE was able to detect the chromosomal rearrangement. Ten percent of the FL cases were negative by all methods tested. In a separate analysis of matched tissue specimens from cases of tumor progression of FL to diffuse lymphoma, PFGE detected a common t(14;18) rearrangement confirming a clonal origin in seven of seven cases, whereas CE detected a rearrangement in only three of seven cases. Overall, PFGE was able to detect a translocation in 8 of 12 cases that were negative by CE and four of eight negative by cytogenetic analysis. In conclusion, PFGE analysis is more comprehensive than CE, PCR, and cytogenetic analysis for the detection of the t(14;18) breakpoint in tissue biopsies of malignant lymphoma.

Abstract

The authors reviewed retrospectively the results of molecular genetic analysis of 175 hematolymphoid lesions to assess the diagnostic utility of genotyping (analysis for immunoglobulin or beta-chain T-cell receptor gene rearrangements). All cases had been genotyped, and most were also immunophenotyped. Cases were assigned to control (90 cases) or problem groups (85 cases), depending on the absence or presence respectively of diagnostic uncertainty remaining after conventional (morphologic and immunophenotypic) analysis. All control cases had unequivocal morphologic features of non-Hodgkin's lymphoma (NHL) and had appropriate lineage-specific gene rearrangements, but immunostaining was almost as sensitive in demonstrating phenotypes that were diagnostically abnormal as well as lineage-specifying. However, genotyping was clearly diagnostically useful in the problem cases, which included a heterogeneous mixture of nodal and extranodal biopsy specimens of malignant (mostly NHL) and benign lesions. Genotyping demonstrated the appropriate absence or presence of gene rearrangements in 58 of 81 problem cases (72%) ultimately diagnosed as benign or malignant respectively, excluding four cases of Hodgkin's disease; in 25 cases (31%) this was judged to be essential to diagnosis. In the remaining problem cases genotyping did not contribute positively to diagnosis, but in no case was genotyping misleading when cautiously interpreted with primary reliance on conventional analysis and with knowledge of known causes of potentially misleading results, both positive and negative. It is concluded that although genotyping is highly sensitive and specific for hematolymphoid neoplasia, it provides little or no benefit in cases that are unequivocally malignant by conventional analysis, in cases of suspected Hodgkin's disease (HD), or for the sole purpose of lineage assignment. However, in cases whose diagnosis is uncertain after conventional analysis, the high yield of useful information from genotyping with careful interpretation warrants its application.

Abstract

The mouse Lyl-1 gene was cloned and shown to consist of four exons with extensive nucleotide and structural homology to the human LYL1 gene. The Lyl-1 gene was localized to the central region of mouse chromosome 8 which defines a new region of synteny with human chromosome 19p. The predicted mouse Lyl-1 protein is 78% identical to human LYL1. The region of highest similarity occurs in the basic DNA binding and helix-loop-helix dimerization motifs which are nearly identical in mouse and man differing by only one conservative amino acid substitution. Expression of the Lyl-1 gene was found to be low in murine spleen and undetectable in other tissues by Northern blot analysis. In lymphoid cell lines, Lyl-1 was expressed in most B lineage cells but downregulated during terminal differentiation and was not expressed in most T lineage cells. In a human T ALL cell line carrying a translocation that juxtaposed LYL1 with the beta TCR gene, the translocated LYL1 gene was transcriptionally active whereas the nontranslocated gene was transcriptionally silent. We conclude that LYL1 has the properties of a lineage- and differentiation-specific HLH protein that contributes to T-cell neoplasia through its deregulated expression following chromosomal translocation.

Abstract

We have studied 36 cases of monocytoid B-cell lymphoma (MBCL). We confirm the predilection for females (30 of 36; ratio, five women to one man). The median age was 65 years (range, 29 to 85 years). Monocytoid B-cell lymphoma characteristically involves peripheral lymph nodes (30 of 36) with a propensity for paraparotid or intraparotid nodes. Salivary glands were affected in five patients. Other extranodal sites of involvement included breast, thyroid, stomach, and soft tissue of chest wall. Eight patients manifested with Sjögren's syndrome, one had systemic lupus erythematosus, one presented initially with Raynaud's phenomenon, and two had a monoclonal gammopathy. "Composite lymphomas" were encountered in seven patients. In addition, association with or progression to a higher-grade lymphoma, ie, mixed small and large cell (one) and large cell (six), was observed in seven patients and was associated with a more aggressive behavior of the lymphoma. Immunohistochemical studies performed on biopsy sections from 20 patients confirmed the B-cell nature of MBCL. An average reactivity of less than 10% of the monocytoid B cells with the proliferation marker Ki-67 was demonstrated, in keeping with the indolent behavior of MBCL. Despite our observation of follicular lymphomas frequently accompanying MBCL, the t(14;18) chromosomal translocation does not appear to play a pathogenetic role for MBCL, as determined by molecular studies for the t(14;18) chromosomal translocation and immunologic studies for the BCL2 protein. Our observations also provide support for the proposal that there is an overlap between MBCL and "MALT lymphomas" (those arising from mucosa-associated lymphoid tissue).

Abstract

The t(1;19)(q23;p13) chromosomal translocation is observed cytogenetically in 25% of children with pre-B-cell acute lymphoblastic leukemia (ALL) and is associated with an adverse treatment outcome. The t(1;19) juxtaposes the E2A gene from chromosome 19 with the PBX1 gene on chromosome 1, leading to the production of fusion transcripts and resultant chimeric proteins that contain the transcriptional-activating motif of E2A and the DNA-binding homeodomain of PBX1. To investigate the molecular nature of E2A/PBX1 fusion in patients with t(1;19) ALL we used an RNA-based polymerase chain reaction (PCR) procedure to amplify a portion of the chimeric transcript. We detected E2A/PBX1 fusion transcripts in cells from 97% (37 of 38) of cases in which the t(1;19) had been observed cytogenetically. Molecular evidence of E2A/PBX1 fusion transcripts was also observed in a patient in whom a t(1;19) was not detected cytogenetically and in one patient with subclinical levels of minimal residual disease before overt clinical relapse. In all PCR-positive cases the junction of E2A and PBX1 coding sequences occurred at precisely the same location as demonstrated by hybridization of PCR products with a fusion site-specific detection oligonucleotide. These findings demonstrate the consistent fusion of E2A and PBX1 coding sequences resulting from t(1;19) and suggest that site-specific fusion of E2A and PBX1 is an important pathogenic event in t(1;19) ALL.

Abstract

The polymerase chain reaction (PCR) was used to detect residual malignant disease before and after ex vivo purging with monoclonal antibodies and complement or immunomagnetic treatment of BM samples contaminated with known numbers of t(14;18)-carrying tumor cells. Sensitivity of the PCR was demonstrated by detecting a specific t(14;18) amplification product in DNA extracted from a preparation consisting of one tumor cell among 10(5) normal cells. When BM contaminated with 1% to 5% t(14;18)-carrying cells from the B-cell lymphoma line SU-DHL-4 was subjected to two rounds of anti-B-cell pool of antibodies and complement (Ab-C) treatment a 3- to 4-log reduction of the pretreatment PCR signal was observed. A similar log-cell kill was detected using an independent clonogenic assay confirming the utility of the PCR approach. BM contaminated with a second B-cell lymphoma cell line, OCI-Ly8, was more resistant because a third cycle of Ab-C treatment was required to obtain a similar reduction in the PCR signal. A similar 4 logs of tumor cell removal was obtained using anti-B-cell antibodies conjugated to magnetic beads. These studies demonstrate that the t(14;18) PCR can be used to detect levels of tumor cells as low as 0.001%. This approach can be used to determine the effectiveness of BM purging in patients undergoing autologous BM transplantation as well as to assess the biologic role of minimal marrow disease.

Abstract

To investigate the relationship of bcr-abl fusion mRNAs with childhood acute lymphoblastic leukemias (ALL), we examined 27 pediatric Philadelphia chromosome (Ph1)-positive acute leukemias using a reverse polymerase chain reaction (PCR) procedure. In cells from 24 leukemias, single bcr-abl PCR products were detected that corresponded to breakpoints in the minor breakpoint cluster region (mbcr in intron 1 of the bcr gene) associated with production of the P190 fusion protein. Cells from the three remaining leukemias contained breakpoints in the major breakpoint cluster region (Mbcr) as shown by PCR and Southern blot analyses. These three leukemias also contained low levels of the mbcr PCR product that may have resulted from alternative splicing of the bcr-abl precursor RNA. A screen of 35 additional leukemias from patients who failed therapy before day 180 (induction failures or early relapses) found one case with unsuccessful cytogenetics to express Mbcr-abl RNA. All four children with Mbcr breakpoints had white blood cell levels in excess of 250,000 at presentation (compared with 2 of 24 with mbcr breakpoints) and two had hematologic and clinical features suggestive of chronic myelogenous leukemias (CML) in lymphoid blast crisis. Our results indicate that in Ph1-positive pediatric leukemias, all 9;22 breakpoints occur in one of the two known breakpoint cluster regions in the bcr gene on chromosome 22. The reverse PCR reliably detected all patients with cytogenetic t(9;22) and is capable of detecting additional Ph1-positive leukemias that are missed by standard cytogenetics. Furthermore, the Mbcr-type breakpoint, associated with production of p210, can be seen in childhood leukemias presenting either as clinical ALL or as apparent lymphoid blast crisis of CML, suggesting that t(9;22) breakpoint locations do not exclusively determine the biologic and clinical features of pediatric Ph1-positive ALL.

Abstract

The 14;18 chromosomal translocation, characteristic of a significant fraction of non-Hodgkin's lymphomas, results from an apparent error in immunoglobulin gene rearrangement. Breakpoints for the 14;18 translocation cluster at defined sites on chromosomes 14 and 18 and this clustering has important implications for molecular diagnostic studies of lymphomas. The major effect of the 14;18 translocation is a transcriptional deregulation of the bcl-2 gene resulting in levels of the protein and mRNA that appear to be inappropriate for B cells at a comparable stage of differentiation. Gene transfer studies have demonstrated that inappropriate bcl-2 expression has subtle effects on cellular growth and survival without overt tumorigenic conversion. Biochemical studies have shown that bcl-2 is an integral membrane protein localized to the cytoplasmic side of cellular membranes, suggestive of a role in signal transduction, but no demonstrable biochemical activity has been reproducibly associated with the protein.

Abstract

We have characterized a chromosomal translocation in a cell line (SU-DUL5) established from a patient with lymphoblastic lymphoma in which the c-myc gene on chromosome 8 was juxtaposed to a t(14;18). Cytogenetic analysis of this cell line showed 14q+, 18q-, and 8p+q+ marker chromosomes in the absence of t(14;18). Genomic Southern blot analysis showed juxtaposition of the immunoglobulin heavy chain joining region (JH) with chromosome 18 near the minor breakpoint cluster region (mcr) of the bcl-2 gene. There was also a rearranged c-myc gene detectable with a 5' c-myc probe. Molecular cloning studies showed that the c-myc gene was joined to chromosome 18 DNA. Nucleotide sequence analysis of cloned breakpoint DNA revealed that the crossover between chromosomes 8 and 18 occurred at the 3' end of the bcl-2 gene resulting in replacement of the bcl-2 gene on the 14q+ chromosome with the c-myc gene. As a result of this translocation the SU-DUL5 cell line contains no detectable bcl-2 mRNA or protein but has abundant levels of c-myc mRNA. Our data suggest that bcl-2 inactivation occurred simultaneously with c-myc translocation in a B cell lymphoblastic lymphoma.

Abstract

Non-random translocation involving the short arm of chromosome 19 are frequently observed in acute leukemias. Recent studies have shown that the 19p13 genes E2A and LYLl, both of which encode helix-loop-helix proteins, lie at two different translocation breakpoints in acute lymphoblastic leukemias (ALL). The E2A gene is involved by the t(1;19)(q23;p13) in acute pre-B-cell leukemias and the LYL1 gene is structurally altered by a t(7;19)(q34;p13) in T-cell ALL. To assess the role of these genes in other leukemia-associated translocations we mapped their locations with respect to the t(11;19)(q23;p13) and t(4;19)(q21;p13) translocation breakpoints carried by T-ALL cell lines SUP-T13 and SUP-T8a, respectively. In situ hybridization studies indicated that the E2A and LYL1 genes are physically distinct from the t(4;19) and t(11;19) breakpoints. Using these and other 19p13 translocation breakpoints as landmarks, we established a partial physical map of 19p: 19pter-E2A-INSR-LYL1-[t(4;19)]-19cen. These data should help guide molecular studies to further characterize 19p13 breakpoints and mapping of genes in this chromosomal region.

Abstract

The t(1;19) chromosomal translocation in acute lymphoblastic pre-B cell leukemias involves the gene E2A for helix-loop-helix (HLH) proteins E12 and E47, ubiquitous transcriptional proteins implicated in the regulation of various lymphoid and nonlymphoid genes. To characterize the molecular features of the t(1;19)(q23;p13) translocation, we molecularly cloned breakpoint DNA from t(1;19)-carrying pre-B cell leukemias. In all cases, breakpoints on chromosome 19 occurred within 2 kb of each other in a single intron of the E2A gene. This clustered arrangement resulted in specific truncation of the E2A gene and transcript, with loss of sequences encoding the basic DNA-binding and HLH dimerization motifs from the derivative 19 chromosome. In contrast, breakpoints on chromosome 1 were distributed over a large region and could not be linked to exonic sequences of the PBX1 gene, although identical chromosome 1 sequences are joined to E2A sequences in 1;19 fusion transcripts. These data show that the 1;19 translocation consistently results in exchange of 3' exons encoding the HLH motifs of E2A with DNA from chromosome 1 to form a fusion gene on the derivative 19 chromosome.

Abstract

A modified polymerase chain reaction (PCR) procedure was used to study the expression of bcr-abl fusion transcripts following allogeneic bone marrow transplantation (BMT) for Philadelphia chromosome (Ph1) positive acute and chronic leukemias. The technique was applied to RNA preparations of peripheral blood and bone marrow cells from 10 patients with chronic myelogenous leukemia (CML) and one patient with acute lymphoblastic leukemia (ALL), all of whom had undergone allogenic BMT and were in clinical and cytogenetic remission. Pre-BMT samples available for eight of 11 patients contained detectable bcr-abl fusion products serving as a baseline for comparison to post-BMT studies. Six patients showed no PCR-detectable bcr-abl transcripts in each of several serial analyses post-BMT (1-36 months post-BMT). The remaining five patients demonstrated various patterns of bcr-abl transcript expression after transplantation. In three patients, bcr-abl transcripts persisted for up to 3 months post-BMT but subsequently were undetectable. Molecular relapse was observed 3 and 6 months post-BMT in the remaining two patients whose earlier post-BMT samples showed no bcr-abl fusion transcripts. No bcr-abl transcripts were detected in subsequent samples from both of these patients 6 months and 1 year post-BMT, respectively. These data confirm that Ph1 carrying cells expressing the bcr-abl fusion mRNA may persist or recur for several months following BMT in the absence of clinical and cytogenetic relapse. The significance of these observations is discussed with respect to results reported recently by others using similar techniques.

SMALL G-PROTEINS ARE EXPRESSED UBIQUITOUSLY IN LYMPHOID-CELLS AND DO NOT CORRESPOND TO BCL-2NATUREMONICA, K., CHENLEVY, Z., Cleary, M. L.1990; 346 (6280): 189-191

Abstract

The bcl-2 gene is consistently associated with t(14; 18) chromosomal translocations observed in a large fraction of human B-cell lymphomas. The t(14; 18) translocation results in deregulated expression of the bcl-2 gene and synthesis of inappropriately high levels of the Bcl-2 protein. Gene transfer studies suggest a role for Bcl-2 in cell survival, growth enhancement and oncogenic transformation. To test the suggestion that GTP-binding by Bcl-2 may mediate its biological effects we characterized the GTP-binding proteins in lymphoid cells expressing Bcl-2. Expression of several small GTP-binding proteins was found to be ubiquitous and did not vary with levels of Bcl-2. By using immunological, electrophoretic and cell-fractionation techniques, we separated Bcl-2 from G proteins of small relative molecular mass (Mr) and showed that it is incapable of binding GTP. Our results show that small Mr G proteins are widely expressed in lymphoid cells and that Bcl-2 is not a novel member of this GTP-binding protein family.

Abstract

The Bcl-2 oncogenic protein was synthesized in vitro and shown to post-translationally integrate asymmetrically into microsomal membranes with no requirement for an amino-terminal signal sequence. Instead, a carboxyl-terminal hydrophobic domain of Bcl-2 served as an insertion sequence essential for membrane assembly since a Bcl-2 mutant lacking this domain completely lost its ability to associate with microsomal membranes. The data demonstrate that Bcl-2 is tightly associated with the lipid bilayer with the nature of an integral membrane protein. The membrane orientation of Bcl-2 was determined using a protease protection assay, which showed that it is predominantly localized to the cytoplasmic face of membranes. A similar type of membrane processing has been shown for cytochrome b5 and also suggested for the viral oncogenic protein polyoma middle-T antigen.

Abstract

Analysis of immunoglobulin (Ig) and T-cell receptor gene rearrangements, using Southern blot hybridization, has been applied to peripheral blood lymphocytes (PBL) in 335 samples from patients with non-Hodgkin's lymphoma. The incidence of circulating lymphoma cells detected by gene rearrangement analyses is related to the histologic subtype, clinical stage of disease, and clinical status. Among 104 patients studied at diagnosis, the incidence of positive analyses was 34% in low-grade lymphoma and only 8% in intermediate-grade lymphoma. Clonal Ig gene rearrangements were detected nearly universally in the small lymphocytic histologic subtype. PBL studies were related to the initial stage of disease: positive studies were seen in 35% of patients with stage IV disease, 29% of patients with stage III disease, and 12% of patients with stages I-II disease. The incidence of PBL rearrangements at the time of disease recurrence in 32 patients requiring cytoreductive therapy was 48%, somewhat greater than at initial diagnosis. A group of patients with low-grade lymphoma, who had treatment deferred after diagnosis or recurrence, was also studied; the incidence of PBL rearrangements was 38% in this population. Among 157 patients clinically free of disease, DNA analyses of the PBL were positive in only 10%. Subsequent relapse of disease in 26 patients was antedated by PBL rearrangement in only one patient. Clonal rearrangements detected in 15 patients have been followed by recurrence of clinical disease in only one patient over a median of 24 months from the time of analysis. The lack of detectable rearrangements in the peripheral blood in the majority of patients may be due to methodology or the biology of the disease. These issues may be further addressed with alternative methods for assessment of minimal disease. However, rigorous testing of any new molecular tool requires an adequate patient population in which disease status is closely monitored over a sufficient period of time.

Abstract

The gene (E2A) for enhancer binding transcription factors E12 and E47 maps to the t(1;19) chromosomal translocation breakpoint in pre-B cell leukemias. Altered E2A transcripts lacking sequences coding for the helix-loop-helix DNA binding motif were detected in several t(1;19)-carrying cell lines. Fusion cDNAs that crossed the t(1;19) breakpoint were cloned and shown to code for an 85 kd protein consisting of the amino-terminal two-thirds of E2A fused to a chromosome 1-derived protein. The fusion protein has the features of a chimeric transcription factor in which the DNA binding domain of E2A is replaced by the putative DNA binding domain of a homeoprotein from chromosome 1 for which the name Prl (pre-B cell leukemia) is proposed. Identical E2A-prl mRNA junctions were detected by PCR in three t(1;19)-carrying cell lines, indicating that the fusion transcripts and predicted chimeric protein are a consistent feature of this translocation.

Abstract

Immunohistochemical and molecular genetic studies were performed on tissues involved by follicular lymphomas that at some point in their course showed a lack of detectable surface or cytoplasmic immunoglobulins (Ig). The variable nature of Ig expression in these lymphomas was evidenced by three tumors biopsied from two different sites that showed an Ig-negative phenotype in one biopsy versus an Ig-positive phenotype in the other. The B lineage derivation of Ig-negative follicular lymphomas was confirmed by the presence of Ig heavy and light chain gene rearrangements in eight of eight lymphomas tested. In a way similar to Ig-expressing follicular lymphomas, the Ig-negative tumors were characterized by bcl-2 gene rearrangements (seven of eight) and overexpression of the Bcl-2 protein (eight out of nine). In two of the three lymphomas with Ig-positive and Ig-negative tumor cell populations, the clonal relationship of the Ig-expressing and nonexpressing cells was established by demonstration of identical t(14; 18) DNA rearrangements. The findings demonstrated that the variability of Ig expression in follicular lymphomas reflects the phenotypic heterogeneity of these tumors and is not a manifestation of separate clonal origins.

Abstract

The gene (E2A) that codes for proteins with the properties of immunoglobulin enhancer binding factors E12/E47 was mapped to chromosome region 19p13.2-p13.3, a site associated with nonrandom translocations in acute lymphoblastic leukemias. The majority of t(1;19)(q23;p13)-carrying leukemias and cell lines studied contained rearrangements of E2A as determined by DNA blot analyses. The rearrangements altered the E2A transcriptional unit, resulting in the synthesis of a transcript larger than the normal-sized E2A mRNAs in one of the cell lines with this translocation. These observations indicate that the gene for a transcription factor is located at the breakpoint of a consistently recurring chromosomal translocation in many acute leukemias and suggest a direct role for alteration of such factors in the pathogenesis of some malignancies.

Abstract

We have characterized a transcription unit at chromosome band 19p13 that lies at the site of a chromosomal translocation breakpoint in T cell acute lymphoblastic leukemia. The lyl-1 gene is structurally altered following a t(7;19) translocation, resulting in its head-to-head juxtaposition with the T cell receptor C beta gene and truncation of lyl-1 RNA. The predicted protein product of the lyl-1 gene contains a potential helix-loop-helix DNA binding motif also found in several proteins involved in the control of cellular proliferation and differentiation: all members of the Myc family, MyoD1, myogenin, the Drosophila achaete-scute, twist, and daughterless proteins, and two recently described immunoglobulin enhancer binding proteins. The implication of lyl-1 in cellular transformation suggests that other proteins containing similar DNA binding motifs may also be involved with neoplastic transformation in various cellular lineages.

Abstract

Cutaneous lymphoid hyperplasia (CLH) is a disorder characterized by the development of one or more skin lesions containing dense lymphoid infiltrates that exhibit the histopathologic features of a benign, reactive process. Nevertheless, some cases have been associated with the subsequent development of clinically overt lymphomas. This suggests that monoclonal populations may exist in some cases of CLH and that these cases may represent a subset more likely to evolve into lymphoma. To determine if such a subset of CLH can be distinguished, Southern blot analysis of DNA was used to study the immunogenotypic features of lesions from 14 patients with clinical, histopathologic, and immunopathologic findings characteristic of CLH. Five cases exhibited detectable clonal rearrangements of immunoglobulin genes. Furthermore, one of these five cases evolved into overt diffuse large cell lymphoma of B cell lineage during a 2-year follow-up of recurrent disease at the original cutaneous site. The immunoglobulin gene rearrangements of this lymphoma were identical to those of the prior CLH lesion. There was no evidence of detectable t(14;18) chromosomal translocations or clonal rearrangements of the beta gene of the T cell receptor in any case. It was concluded that CLH can be divided into two subsets based on the presence or absence of a clonal B cell population, and that overt lymphoma can arise from the former subset and contain the same B cell clone identified in the pre-existent CLH lesion.

Abstract

We report a new methodology for the long-term growth of malignant T-lymphoblasts from patients with T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LL). When malignant cells were cultured in the presence of insulin-like growth factor I under hypoxic conditions, cellular proliferation occurred that resulted in the establishment of immortal cell lines from ten of 12 patient tumors. Authenticity of each cell line was verified by a direct comparison of the immunophenotype, karyotype, and immunogenotype with the patient's tumor cells. This improved method of cell culture permits frequent establishment of cell lines from patients with T-ALL/T-LL, thereby aiding in analysis of thymocyte transformation and neoplasia.

Abstract

A majority of t(14;18) translocations have been shown to cluster at one of two sites on chromosome 18, called the major breakpoint region (mbr) or the minor cluster region, (mcr), which map within or flanking the bcl-2 proto-oncogene, respectively. We have determined the nucleotide sequence for a portion of the mcr, and constructed oligonucleotides that were used to perform the polymerase chain reaction (PCR) in conjunction with universal immunoglobulin primers to specifically amplify t(14;18) breakpoints in DNA obtained from follicular lymphomas. Eight of ten breakpoints that were detectable on Southern blots using DNA probes for the mcr could be detected due to specific amplification by the PCR technique using an mcr-specific primer. Direct nucleotide sequencing of the enzymatically amplified DNAs showed that the breakpoints clustered within a 500 nucleotide region, and five occurred within three nucleotides of each other. These data show a remarkable clustering of some t(14;18) breakpoints at a site on chromosome 18, at least a 30-kb distance from the bcl-2 gene. Our findings also indicate that mcr-specific primers may be used in conjunction with previously described mbr-specific primers in a highly sensitive DNA amplification technique to detect a large fraction of t(14;18) breakpoints.

Abstract

The vast majority of mature T cells express the alpha, beta TCR and use this receptor to recognize Ag in association with MHC determinants. Recent reports indicate that a small subpopulation of CD3+ T cells does not express alpha, beta TCR and lacks CD4 and CD8 molecules, but expresses another TCR heterodimer designated gamma, delta. To date, however, there are very few examples of gamma, delta TCR-bearing T cells which have Ag-specific functions and the precise role of the gamma, delta TCR remains unknown. In an attempt to explore the function of the gamma, delta TCR in man, we generated a panel of CD3+, CD4-, CD8- T cell clones from cultures of normal T cells stimulated with allogeneic lymphoblastoid cell line (LCL). All of the clones stained with mAb to gamma, delta TCR determinants, and Northern blot analysis of total cellular RNA revealed mature gamma- and delta-chain transcripts and immature beta-chain transcript. The clones displayed cytolytic activity against their specific stimulating LCL, but not irrelevant LCL, and killing was inhibited by a mAb to HLA class I (HLA-A,B,C) determinants, suggesting that these cells recognized class I MHC antigenic determinants on specific stimulator/target cells. Cytolysis by these clones was markedly inhibited by low concentrations (0.00001 to 0.001 microgram/ml) of mAb to CD3 or TCR delta-chain, but high concentrations (0.1 to 20 micrograms/ml) of the same antibodies enhanced cytotoxicity. When absorbed to plastic tissue culture wells, anti-CD3 mAb stimulated the clones to proliferate. These results indicate that alloantigen specific, gamma, delta TCR+, CD4-, CD8- T cell clones can be reproducibly generated in vitro, and that the gamma, delta TCR plays a direct role in the specific recognition and cytolysis displayed by these clones.

Abstract

We have identified a 24-kilodalton protein that is the product of the human bcl-2 gene, implicated as an oncogene because of its presence at the site of t(14;18) translocation breakpoints. The Bcl-2 protein was detected by specific, highly sensitive rabbit antibodies and was shown to be present in a number of human lymphoid cell lines and tissues, as well as in mouse B cells transfected with a bcl-2 cDNA construct. Characterization of the Bcl-2 protein demonstrated that it has a lipophilic nature and is associated with membrane structures, probably by means of its hydrophobic carboxy-terminal membrane-spanning domain. In t(14;18)-carrying cell lines, the protein is predominantly localized to the perinuclear endoplasmic reticulum, with a minor fraction in the plasma membrane. These properties, together with the observations that Bcl-2 does not have a characteristic signal peptide and is not glycosylated, suggest that it is an integral-membrane protein that spans the bilayer at its C-terminal hydrophobic region but is exposed only at the cytoplasmic surface. The relative abundance of the Bcl-2 protein in various human lymphoid cell lines correlated with transcription of the bcl-2 gene. The protein was abundant in all t(14;18)-carrying cell lines and lymphomas and was also found at lower levels in pre-B-cell lines and nonmalignant lymphoid tissues that do not carry t(14;18) translocations. These results suggest that the Bcl-2 protein is functional in normal B lymphocytes and that a quantitative difference in its expression may play a role in the pathogenesis of lymphomas carrying the t(14;18) translocation.

Abstract

The clonal composition of various transplant-associated lymphoproliferations was assessed by means of Southern blot hybridizations using a DNA probe specific for the fused termini of the Epstein-Barr virus (EBV) genome. A single clonal band representing the joined EBV genomic termini was detected in most biopsies, demonstrating the presence of a monoclonal expansion of B lymphocytes carrying EBV DNA. Different configurations of immunoglobulin gene rearrangements and fused EBV genomic termini were frequently observed in tissues from different biopsy sites in individual patients, confirming the multiclonal origins for these lymphomas. In rare specimens, multiple forms of the joined termini were detected within individual lesions, which appeared polymorphous by histologic methods of analysis and polyclonal by immunologic and immunogenetic methods of analysis. These studies confirm that there is a spectrum of EBV-associated disorders of varying clonal composition that may arise in immunosuppressed organ-allograft recipients. The data are consistent with the proposal that the lymphoproliferations initiate as polyclonal expansions of EBV-carrying B cells, which progress to multiclonal lymphomas in most patients. Detection of homogeneous episomal EBV DNA in most lesions supports a primary role for the virus in the pathogenesis of these disorders.

Abstract

For many non-Hodgkin's lymphomas, the bcl-2 gene has been implicated as a likely proto-oncogene, since it is consistently located at or near the breakpoint sites of t(14;18) chromosomal translocations. To define the role of the protein product of the bcl-2 gene in lymphoid cancers, we used anti-bcl-2 antibodies to perform immunohistochemical studies of frozen sections of 136 tissue specimens affected by lymphoma or non-neoplastic lymphoid disorders. Immunoreactive bcl-2 protein was observed in the neoplastic cells in almost all the follicular lymphomas, whereas no bcl-2 protein was detected in follicles affected by non-neoplastic processes or in normal lymphoid tissue. Every tumor with molecular-genetic evidence of t(14;18) translocation expressed detectable levels of bcl-2 protein, regardless of whether the breakpoint was located in or at a distance from the bcl-2 gene. These data show consistent expression of a proto-oncogenic protein in a large proportion of non-Hodgkin's lymphomas and provide further support of a role for bcl-2 in the pathogenesis of all lymphomas with the t(14;18) karyotypic abnormality. Increased expression of bcl-2 after t(14;18) translocations may be a specific marker for B-cell cancers, and demonstration of the protein with use of anti-bcl-2 antibodies could be useful in the diagnosis of many non-Hodgkin's lymphomas.

Abstract

Immunoglobulin (Ig) or idiotype (Id) is a tumor-specific target in those B cell malignancies that express this molecule on their surface. We explored the biology of B cell acute lymphoblastic leukemia (B cell ALL) using Id as a tumor marker. In this report we describe the development of anti-Id monoclonal antibodies (MAB) for two children with B cell ALL. These reagents were used retrospectively to study tumor kinetics and to detect residual disease after chemotherapy. In both cases serum Id values were strikingly high at diagnosis (1.2 mg/mL and 10.8 mg/mL), suggesting that the tumor cells were relatively mature B cells capable of significant antibody production. In both patients the serum Id levels fell with the institution of therapy and confirmed that the patients were in remission. Increasing serum Id predicted relapse four months before conventional methods in patient 1, and Id proved to be a more sensitive measure of tumor burden than Southern blot analysis of rearranged Ig genes in bone marrow samples. Surprisingly, low levels of Id were redetected in the second patient just before completing therapy and have persisted for over a year despite the absence of clinical evidence of recurrent disease. Thus, serum Id levels reflect tumor burden during initial therapy but may not necessarily predict tumor progression after a complete clinical remission.

Abstract

DNA spanning a t(7;19) chromosomal translocation breakpoint was isolated from the human T cell line SUP-T7 established from an acute lymphoblastic leukemia. Nucleotide sequence analysis showed that the point of crossover on chromosome 7 occurred immediately adjacent to joining segment J beta 1.1 within the TCR-beta gene, suggesting that this translocation resulted from an error in TCR gene rearrangement. On chromosome 19, the translocation occurred within a previously uncharacterized transcriptional unit for which we propose the name lyl-1. An approximately 1.5-kb RNA is transcribed from this gene in a wide variety of hematolymphoid cell lines. The t(7;19) results in truncation of the lyl-1 gene and production of abnormal-sized RNAs, suggesting a role for lyl-1 in the pathogenesis of this leukemia.

Abstract

To investigate the possible relatedness of the subpopulations that make up so-called biclonal lymphomas, we examined five bigenotypic and biphenotypic follicular lymphomas using DNA probes specific for the t(14;18) chromosomal translocation, which is a characteristic feature of these neoplasms. On Southern blot analysis, both subpopulations from four of five lymphomas contained comigrating t(14;18) DNA rearrangements, confirming the single cell origins for these neoplasms. No comigrating t(14;18) DNA rearrangements were observed in the fifth lymphoma, but nucleotide sequence analysis of cloned, breakpoint DNA showed identical t(14;18) crossovers in the two subpopulations. The migration differences of both the Ig and chromosome 18 DNA rearrangements were shown to result from somatically acquired mutations of the Ig genes from the fifth lymphoma. These studies indicate that Ig gene rearrangements and idiotope expression are not consistently stable clonal markers since they are subject to variability as a result of somatic mutation. Although translocated chromosome 18 DNA rearrangements are more reliable, they may also vary among cells of some tumors since somatic mutation can affect, as well, DNA of translocated alleles in follicular lymphomas.

Abstract

One of the most common karyotypic abnormalities is the t(14;18) translocation, which is found in many lymphomas that have a characteristic follicular morphology. Recent molecular studies have shown that this chromosomal translocation results in the juxtaposition of the candidate proto-oncogene bcl-2 (B-cell leukemia-lymphoma) on chromosome 18 with the immunoglobulin heavy-chain locus on chromosome 14. However, because performing accurate cytogenetic studies in solid hematolymphoid neoplasms is difficult, knowledge of the prevalence of the t(14;18) translocation and, by association, the extent of bcl-2 involvement in human lymphomas is limited. We used a number of chromosome-18 DNA probes to analyze various subtypes of Hodgkin's and non-Hodgkin's lymphomas and test for structural abnormalities near or within the bcl-2 gene. Molecular features of the t(14;18) translocation were found in virtually all follicular neoplasms and about 28 percent of diffuse large-cell lymphomas. No changes in bcl-2 were found in several other subtypes of Hodgkin's and non-Hodgkin's lymphomas, including those previously suggested to originate from follicular-center cells and those about which cytogenetic data have been difficult to obtain. Our findings suggest a close pathogenetic relation between bcl-2 and a large group of non-Hodgkin's lymphomas, both with and without a follicular morphology. The methods employed in this study may be useful in improving the accuracy of diagnosis and subclassification of malignant lymphomas.